Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system.

The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.

Using Radiomics to Differentiate Brain Metastases From Lung Cancer Versus Breast Cancer, Including Predicting Epidermal Growth Factor Receptor and human Epidermal Growth Factor Receptor 2 Status.

OBJECTIVE: We evaluated the feasibility of using multiregional radiomics to identify brain metastasis (BM) originating from lung adenocarcinoma (LA) and breast cancer (BC) and assess the epidermal growth factor receptor (EGFR) mutation and human epidermal growth factor receptor 2 (HER2) status. METHODS: Our experiment included 160 patients with BM originating from LA (n = 70), BC (n = 67), and other tumor types (n = 23), between November 2017 and December 2021. All patients underwent contrast-enhanced T1- and T2-weighted magnetic resonance imaging (MRI) scans. A total of 1967 quantitative MRI features were calculated from the tumoral active area and peritumoral edema area and selected using least absolute shrinkage and selection operator regression with 5-fold cross-validation. We constructed radiomic signatures (RSs) based on the most predictive features for preoperative assessment of the metastatic origins, EGFR mutation, and HER2 status. Prediction performance of the constructed RSs was evaluated based on the receiver operating characteristic curve analysis. RESULTS: The developed multiregion RSs generated good area under the receiver operating characteristic curve (AUC) for identifying the LA and BC origin in the training (AUCs, RS-LA vs RS-BC, 0.767 vs 0.898) and validation (AUCs, RS-LA vs RS-BC, 0.778 and 0.843) cohort and for predicting the EGFR and HER2 status in the training (AUCs, RS-EGFR vs RS-HER2, 0.837 vs 0.894) and validation (AUCs, RS-EGFR vs RS-HER2, 0.729 vs 0.784) cohorts. CONCLUSIONS: Our results revealed associations between brain MRI-based radiomics and their metastatic origins, EGFR mutations, and HER2 status. The developed multiregion combined RSs may be considered noninvasive predictive markers for planning early treatment for BM patients.

Radiomics evaluates the EGFR mutation status from the brain metastasis: a multi-center study.

Objective.To develop and externally validate habitat-based MRI radiomics for preoperative prediction of the EGFR mutation status based on brain metastasis (BM) from primary lung adenocarcinoma (LA).Approach.We retrospectively reviewed 150 and 38 patients from hospital 1 and hospital 2 between January 2017 and December 2021 to form a primary and an external validation cohort, respectively. Radiomics features were calculated from the whole tumor (W), tumor active area (TAA) and peritumoral oedema area (POA) in the contrast-enhanced T1-weighted (T1CE) and T2-weighted (T2W) MRI image. The least absolute shrinkage and selection operator was applied to select the most important features and to develop radiomics signatures (RSs) based on W (RS-W), TAA (RS-TAA), POA (RS-POA) and in combination (RS-Com). The area under receiver operating characteristic curve (AUC) and accuracy analysis were performed to assess the performance of radiomics models.Main results.RS-TAA and RS-POA outperformed RS-W in terms of AUC, ACC and sensitivity. The multi-region combined RS-Com showed the best prediction performance in the primary validation (AUCs, RS-Com versus RS-W versus RS-TAA versus RS-POA, 0.901 versus 0.699 versus 0.812 versus 0.883) and external validation (AUCs, RS-Com versus RS-W versus RS-TAA versus RS-POA, 0.900 versus 0.637 versus 0.814 versus 0.842) cohort.Significance.The developed habitat-based radiomics models can accurately detect the EGFR mutation in patients with BM from primary LA, and may provide a preoperative basis for personal treatment planning.

Glucose-Responsive Microspheres as a Smart Drug Delivery System for Controlled Release of Insulin.

BACKGROUND AND OBJECTIVES: Diabetes mellitus, a disease of glucose regulation, has become one of the most common medical problems in the world. At present, alternative therapy for diabetes has, to a large extent, been widely concerned with the improvement of treatment efficacy. The aims of this study were to characterize and evaluate the surface morphology of the novel glucose-responsive injectable microspheres containing insulin, along with their in vitro release and in vivo efficacy. METHODS: In this study, glucose-responsive microspheres as an emerging smart drug delivery system for controlled release of insulin were developed by an improved water-in-oil-in-water (W/O/W) double emulsion preparation method. Here, methoxypolyethylene glycol-hydrazone-4-methoxypolyethylene glycol benzoate (mPEG-Hz-mPEG4AB) was synthesized as a pH-responsive carrier. RESULTS: The microspheres had a good spherical structure with a particle size of 5 ~ 10 µm. Approximately 61% of insulin was released in 15 h under a high glucose environment but was barely released within the normal glucose range in in vitro studies. After a subcutaneous injection of insulin microspheres in rats, blood glucose levels rapidly decreased within 2 h and could be maintained for 2 days in the normal range. Histopathological evaluation indicated that the microspheres were almost non-irritating. CONCLUSIONS: The pH-responsive mPEG-Hz-mPEG4AB could be used as an efficient insulin microsphere carrier, and the optimized microspheres had good morphology and sustained hypoglycemic effect.