CRISPR-engineered T cells in patients with refractory cancer.

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRß (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

Association of Coronary Artery Calcification and Mortality in the National Lung Screening Trial: A Comparison of Three Scoring Methods.

PURPOSE: To evaluate three coronary artery calcification (CAC) scoring methods to assess risk of coronary heart disease (CHD) death and all-cause mortality in National Lung Screening Trial (NLST) participants across levels of CAC scores. MATERIALS AND METHODS: The NLST was approved by the institutional review board at each participating institution, and informed consent was obtained from all participants. Image review was HIPAA compliant. Five cardiothoracic radiologists evaluated 1575 low-dose computed tomographic (CT) scans from three groups: 210 CHD deaths, 315 deaths not from CHD, and 1050 participants who were alive at conclusion of the trial. Radiologists used three scoring methods: overall visual assessment, segmented vessel-specific scoring, and Agatston scoring. Weighted Cox proportional hazards models were fit to evaluate the association between scoring methods and outcomes. RESULTS: In multivariate analysis of time to CHD death, Agatston scores of 1-100, 101-1000, and greater than 1000 (reference category 0) were associated with hazard ratios of 1.27 (95% confidence interval: 0.69, 2.53), 3.57 (95% confidence interval: 2.14, 7.48), and 6.63 (95% confidence interval: 3.57, 14.97), respectively; hazard ratios for summed segmented vessel-specific scores of 1-5, 6-11, and 12-30 (reference category 0) were 1.72 (95% confidence interval: 1.05, 3.34), 5.11 (95% confidence interval: 2.92, 10.94), and 6.10 (95% confidence interval: 3.19, 14.05), respectively; and hazard ratios for overall visual assessment of mild, moderate, or heavy (reference category none) were 2.09 (95% confidence interval: 1.30, 4.16), 3.86 (95% confidence interval: 2.02, 8.20), and 6.95 (95% confidence interval: 3.73, 15.67), respectively. CONCLUSION: By using low-dose CT performed for lung cancer screening in older, heavy smokers, a simple visual assessment of CAC can be generated for risk assessment of CHD death and all-cause mortality, which is comparable to Agatston scoring and strongly associated with outcome.

Conformational analysis of r207910, a new drug candidate for the treatment of tuberculosis, by a combined NMR and molecular modeling approach.

R207910 is an enantiomeric compound from a new class of antimycobacterial agents, the diarylquinolines [Science; 307:223 (2005)]. As enantiospecific interaction is required for biologic activity, we have undertaken a combined nuclear magnetic resonance and molecular modeling study to gain new insights into its conformation in solution and its absolute configuration. A conformational analysis using a Monte-Carlo method has been performed on each of the four possible stereomers of this compound leading to the identification of their most stable conformation. Additional ab initio calculation was performed with emphasis on the strength of the observed intramolecular hydrogen bond. Simultaneously, a complete structural identification has been carried out by a set of monodimensional and bidimensional (1)H-(13)C-NMR experiments. Determination of inter-proton distances has been achieved by a series of (1)H-(1)H ROESY NMR experiments with different mixing times followed by a volume quantification of the correlations peaks. These experimental data were compared with the theoretical distances obtained from the conformational analysis. The remarkable match shows that R207910 adopts one of the low-energy conformations predicted by molecular modeling and belongs to the (RS, SR) couple of diastereoisomers. A posteriori validation of our approach has been performed by X-ray structure determination that concluded for the RS configuration.

A NMR and theoretical study of the aggregates between alkyllithium and chiral lithium amides: control of the topology through a single asymmetric center.

The complexes between methyllithium and chiral 3-aminopyrrolidine (3-AP) lithium amides bearing a second asymmetric center on their lateral amino group were studied using multinuclear ((1)H, (6)Li, (13)C, (15)N) low-temperature NMR spectroscopies in tetrahydrofuran-d(8). The results indicate that lithium chelation forces the pyrrolidine ring of the 3-AP to adopt a norbornyl-like conformation and that robust 1:1 noncovalent complexes between methyllithium and 3-AP lithium amides form in the medium. A set of (1)H-(1)H and (1)H-(6)Li NMR cross-coupling correlations shows that the binding of methyllithium can take place along the "exo" or the "endo" face of this puckered structure, depending on the relative configuration of the lateral chiral group. This aggregation step renders the nitrogen of the 3-amino group chiral, the "exo" and "endo" topologies corresponding to the (S) and (R) configurations, respectively, of this atom. Density functional theory calculations show that the "exo" and "endo" arrangements are, for both diastereomers, almost isoenergetic even when solvent is taken into account. This result suggests that the formation of the mixed aggregates is under strict kinetic control. A relationship between the topology of these complexes and the sense of induction in the enantioselective alkylation of aromatic aldehydes by alkyllithiums is proposed.