Lipid nanoparticles with PEG-variant surface modifications mediate genome editing in the mouse retina.

Ocular delivery of lipid nanoparticle (LNPs) packaged mRNA can enable efficient gene delivery and editing. We generated LNP variants through the inclusion of positively charged-amine-modified polyethylene glycol (PEG)-lipids (LNPa), negatively charged-carboxyl-(LNPz) and carboxy-ester (LNPx) modified PEG-lipids, and neutral unmodified PEG-lipids (LNP). Subretinal injections of LNPa containing Cre mRNA in the mouse show tdTomato signal in the retinal pigmented epithelium (RPE) like conventional LNPs. Unexpectedly, LNPx and LNPz show 27% and 16% photoreceptor transfection, respectively, with striking localization extending from the photoreceptor synaptic pedicle to the outer segments, displaying pan-retinal distribution in the photoreceptors and RPE. LNPx containing Cas9 mRNA and sgAi9 leads to the formation of an oval elongated structure with a neutral charge resulting in 16.4% editing restricted to RPE. Surface modifications of LNPs with PEG variants can alter cellular tropism of mRNA. LNPs enable genome editing in the retina and in the future can be used to correct genetic mutations that lead to blindness.

Lipid Nanoparticle-Enabled Intracellular Delivery of Prime Editors.

Prime editing is an advanced gene editing platform with potential to correct almost any disease-causing mutation. As genome editors have evolved, their size and complexity have increased, hindering delivery technologies with low-carrying capacity and endosomal escape. We formulated an array of lipid nanoparticles (LNPs) containing prime editors (PEs). We were able to encapsulate PEs in LNPs and confirmed the presence of PE mRNA and two different guide RNAs using HPLC. In addition, we developed a novel reporter cell line for rapid identification of LNPs suited for prime editing. A 54% prime editing rate was observed with enhanced LNPs (eLNPs) containing the cholesterol analog ß-sitosterol at optimal ratios of RNA cargoes. eLNPs displayed a polyhedral morphology and a more fluid membrane state that led to improved endosomal escape, eventually causing onset of editing within 9 h and reaching maximum efficiency after 24 h. Hence, PEs delivered using LNPs can propel a new wave of therapies for many additional targets potentially enabling a range of new applications.

Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates.

Lipid nanoparticle (LNP)-based mRNA delivery holds promise for the treatment of inherited retinal degenerations. Currently, LNP-mediated mRNA delivery is restricted to the retinal pigment epithelium (RPE) and Müller glia. LNPs must overcome ocular barriers to transfect neuronal cells critical for visual phototransduction, the photoreceptors (PRs). We used a combinatorial M13 bacteriophage-based heptameric peptide phage display library for the mining of peptide ligands that target PRs. We identified the most promising peptide candidates resulting from in vivo biopanning. Dye-conjugated peptides showed rapid localization to the PRs. LNPs decorated with the top-performing peptide ligands delivered mRNA to the PRs, RPE, and Müller glia in mice. This distribution translated to the nonhuman primate eye, wherein robust protein expression was observed in the PRs, Müller glia, and RPE. Overall, we have developed peptide-conjugated LNPs that can enable mRNA delivery to the neural retina, expanding the utility of LNP-mRNA therapies for inherited blindness.

Mining LTR-retrotransposon genes for mRNA delivery.

RNA-based therapeutics commonly use exogenous components to shuttle their cargo, leading to nonselectivity or immunogenicity. Segel et al. have elucidated an endogenous modular retroviral-like delivery system capable of encapsulating mRNA, which elicits effective transport inside cells, priming the development of endogenous vectors for gene delivery for amelioration of disease.

Flow-down of safety from general contractors to subcontractors working on commercial construction projects.

Small and medium construction firms have high injury rates but lack resources to establish and maintain effective safety programs. General contractors with exemplary safety programs may serve as intermediaries to support development of smaller firm's safety programs. The purpose of this study was to examine the flow-down influence of general contractors' safety programs on the safety climate and safety behaviors among workers employed by small and medium sized subcontractors. This study collected workers' perception of safety climate and safety behaviors while working on projects with general contractors with exemplary safety programs and other general contractors. We also documented the safety program policies and practices of subcontractors, and recorded the changes in safety policies required from general contractors with exemplary programs. We examined differences in safety program changes between small and medium sized subcontractors. Results showed workers perceived general contractors with exemplary safety programs had stronger safety climates than other general contractors. Smaller subcontractors had less robust safety programs with fewer safety elements than larger subcontractors, and therefore, many of the smaller subcontractors had to adopt more safety policies and practices to work for general contractors than large subcontractors. These findings suggest that general contractors with robust safety programs can serve as intermediaries and influence the development of the safety programs of small sized firms. Future work will need to determine if smaller firms eventually adopt safety policies and practices as part of their permanent safety program.

The association between subcontractor safety management programs and worker perceived safety climate in commercial construction projects.

PROBLEM: Safety management programs (SMPs) are designed to mitigate risk of workplace injuries and create a safe working climate. The purpose of this project was to evaluate the relationship between contractors' SMPs and workers' perceived safety climate and safety behaviors among small and medium-sized construction subcontractors. METHODS: Subcontractor SMP scores on 18 organizational and project-level safety items were coded from subcontractors' written safety programs and interviews. Workers completed surveys to report perceptions of their contractor's safety climate and the safety behaviors of coworkers, crews, and themselves. The associations between SMP scores and safety climate and behavior scales were examined using Spearman correlation and hierarchical linear regression models (HLM). RESULTS: Among 78 subcontractors working on large commercial construction projects, we found striking differences in SMP scores between small, medium, and large subcontractors (p < 0.001), related to a number of specific safety management practices. We observed only weak relationships between SMP scales and safety climate scores reported by 746 workers of these subcontractors (ß = 0.09, p = 0.04 by HLM). We saw no differences in worker reported safety climate and safety behaviors by contractor size. DISCUSSION: SMP only weakly predicted safety climate scales of subcontractors, yet there were large differences in the quality and content of SMPs by size of employers. SUMMARY: Future work should determine the best way to measure safety performance of construction companies and determine the factors that can lead to improved safety performance of construction firms. Practical applications: Our simple assessment of common elements of safety management programs used document review and interviews with knowledgeable representatives. These methods identified specific safety management practices that differed between large and small employers. In order to improve construction safety, it is important to understand how best to measure safety performance in construction companies to gain knowledge for creating safer work environments.

Thrombocytosis as a predictor of distant recurrence in patients with rectal cancer.

BACKGROUND AND AIMS: Thrombocytosis is frequently observed in patients with malignancy. We undertook this study to determine the prognostic value of thrombocytosis in patients with rectal cancer. METHODS: We performed a retrospective study of patients undergoing low anterior resection for rectal cancer between January 2000 and March 2007. Preoperative platelet count was measured before surgery. Postoperative platelets were determined 1 month after surgery. Two-tailed p values <0.05 were considered statistically significant. RESULTS: One hundred sixty three patients with rectal cancer were included in the study. Preoperative platelet count >350,000 was found in 8% of patients. Postoperative platelet count >350,000 was found in 6% of patients. Distant metastases were found in 17 patients (10.4%). Significant variables in the multivariate analyses were preoperative platelets >350,000 (p = 0.001), postoperative platelets >350,000 (p = 0.002), carcinoembryonic antigen >13 ng/dL (p = 0.003). Patients with preoperative platelet count <350,000 showed a 5-year survival rate of 81%, whereas patients with platelet count >350,000 had a 25-month survival [95% confidence interval (CI): 20-26]; p <0.001. Patients with postoperative platelets <350,000 showed a 5-year survival rate of 80%, whereas patients with platelets >350,000 showed a 3-year survival rate of 37.5% (p <0.05). CONCLUSIONS: Pre- or postoperative platelet count >350,000 is associated with poor survival in patients with rectal cancer. The measurement of platelets is a clinical marker useful to define the prognosis for patients with rectal cancer.