Virus-free CRISPR knock-in of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells.

Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR/Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knock-in of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-Seq, in-out PCR, and next generation sequencing. KLRC1 -GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2 + human melanoma cells. Notably, KLRC1 -GD2 CAR NK cells overcome HLA-E-based inhibition by HLA-E-expressing, GD2 + melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR/Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E + solid tumors.

Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain. METHODS: Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG™ xenograft neuroblastoma model. RESULTS: In comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model. CONCLUSIONS: This study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.

Evaluating pain, sedation, and delirium in the neurologically critically ill-feasibility and reliability of standardized tools: a multi-institutional study.

OBJECTIVES: To assess the feasibility and reliability of systematic evaluations of analgesia, sedation level, and delirium features in the neurologically critically ill and to determine whether delirium features are linked to clinical outcomes in this population. DESIGN: Multicentered prospective observational study. SETTING: Neurological, Neurosurgical, Neurosciences or Surgical Trauma ICUs from three hospitals (two in Canada and one in the United States). PATIENTS: A convenience sample of adult NICU or neurologic, neurosurgical, neurosciences, or surgical trauma ICU patients admitted for greater than 12 hours from November 2011 to April 2012. INTERVENTIONS: Systematic assessments were simultaneously and independently performed by a neurologist, intensivists, or trauma surgeon, and a nurse in three multispecialty ICUs. Pain was evaluated with the numeric rating scale or behavioral pain scale. Sedation was assessed using the Richmond Agitation-Sedation Scale. Patients with Richmond Agitation-Sedation Scale greater than or equal to -4 were screened for features of delirium with the Intensive Care Delirium Screening Checklist. Intraclass correlation coefficient was used to evaluate inter-rater reliability between the nurse and the physician for pain and sedation scales, and the kappa coefficient was calculated for concordance of the Intensive Care Delirium Screening Checklist items. MEASUREMENTS AND MAIN RESULTS: 151 patients had 439 assessments. Pain and sedation were always assessable with excellent inter-rater reliability (numeric rating scale intraclass correlation coefficient, 0.92; behavior pain scale intraclass correlation coefficient, 0.83; and Richmond Agitation-Sedation Scale intraclass correlation coefficient, 0.92). Patients were sufficiently alert for delirium screening 3/4 of the time; Intensive Care Delirium Screening Checklist items had good concordance (kappa coefficients between 0.58 and 0.91 for the eight Intensive Care Delirium Screening Checklist items). Nonevaluable items were most often orientation, hallucinations, and speech or mood content. Furthermore, each additional Intensive Care Delirium Screening Checklist item present in proportion to the total evaluable Intensive Care Delirium Screening Checklist score was associated with a 10% increase in ICU length of stay. CONCLUSIONS: Pain and sedation can be systematically assessed in the neurologically critically ill; the majority can also be screened for delirium features with excellent inter-rater reliability. Increased proportion of Intensive Care Delirium Screening Checklist items is associated with worse outcomes.

Work- and nonwork-related factors associated with PACU nurses' fatigue.

The purpose of this study was to describe postanesthesia care unit (PACU) nurses' fatigue and link fatigue levels to work- and nonwork-related factors. The study design is a pilot study using a descriptive correlation design. Participants used a 4-week daily diary to record hours worked, breaks taken, and sleep patterns and completed the Occupational Fatigue Exhaustion Recovery Scale (OFER). The sample included 20 experienced (17.3 ± 9.5 years) nurses. Only 4% reported no breaks during their shift. Median sleep time was 6 hours 40 minutes. OFER scores were acute fatigue (66.5 ± 19.3), intershift fatigue (52 ± 18.6), and chronic fatigue (35.7 ± 17.2). In conclusion, acute fatigue scores reflect the challenges of working in the PACU. Despite high acute fatigue scores, intershift fatigue scores reflected recovery and chronic fatigue scores were low. Fatigue reduction strategies may account for these results including processes to ensure breaks are taken, use of a flex shift nurse to prevent shift overruns, and reduction of the number of three consecutive 12-hour shifts.

Impact of a delirium screening tool and multifaceted education on nurses' knowledge of delirium and ability to evaluate it correctly.

BACKGROUND: The impact of using a validated delirium screening tool and different levels of education on surgical-trauma intensive care unit (STICU) nurses' knowledge about delirium is unclear. OBJECTIVES: To measure the impact of using the Intensive Care Delirium Screening Checklist (ICDSC), with or without a multi-faceted education program, on STICU nurses' knowledge and perceptions of delirium and their ability to evaluate it correctly. METHODS: The knowledge and perceptions of subject nurses about delirium, and agreement between the independent assessments of delirium by the subject nurse and by a validated judge (who always used the ICDSC), were compared across 3 phases. Phase 1: No delirium screening tool and no education. Phase 2: ICDSC and minimal education (ie, ICDSC validation study only). Phase 3: ICDSC and multifaceted education (ie, pharmacist-led didactic lecture, Web-based module, and nurse-led bedside training). RESULTS: Nurses' knowledge (mean [SD] score out of 10 points) was similar (P = .08) in phase 1 (6.1 [1.4]) and phase 2 (6.5 [1.4]) but was greater (P = .001) in phase 3 (8.2 [1.4]). Agreement between nurses and the validated judge in the assessment of delirium increased from phase 1 (κ = 0.40) to phase 2 (κ = 0.62) to phase 3 (κ = 0.74). Nurses perceived use of the ICDSC as improving their ability to recognize delirium. CONCLUSIONS: Use of a multifaceted education program improves both nurses' knowledge about delirium and their perceptions about its recognition. Implementation of the ICDSC improves the ability of STICU nurses to evaluate delirium correctly.