A hybridized mechano-electroporation technique for efficient immune cell engineering.

Immune cell engineering, which involves genetic modification of T cells, natural killer cells, and macrophages, is shifting the paradigm in immunotherapy for treating hematologic malignancies. These modified cells can be viewed as living drugs and offer advantages, including dynamic functionality, active local trafficking, and boosting the immune system while recognizing and eliminating malignant cells. Among the current technologies employed for the modification of immune cell functions, electroporation stands as a predominant approach, but it suffers from heterogeneity arising from the treatment of a bulk population of immune cells during the manufacturing procedures. To address this challenge of the field, here we present a hybrid approach to induce consecutive gentle mechanical and electric shocks. This approach enhances the treatment homogeneity and improves outcomes in difficult-to-load immune cells. The hybrid approach aims to enhance the treatment homogeneity by passing individual immune cells through a microengineered filter membrane with micropores smaller than the cell diameter. This facilitates the creation of transient pores in the cell membrane, followed by efficient delivery of biomolecules through the complementary use of a gentle electric shock. Using this hybrid mechano-electroporation (HMEP) system, we could successfully deliver fluorescein isothiocyanate (FITC) dextran molecules from the smallest (4 kDa) to the largest (2000 kDa) size and EGFP expressing plasmid DNA into different immune cell types. We also provide insight into the delivery performance of the HMEP system in comparison with the benchtop electroporation since both methods hinge on membrane disruption as their permeabilization mechanism. Immune cells treated with the HMEP protocol demonstrated higher delivery efficiencies while maintaining cell viability compared to those experiencing conventional electroporation. Therefore, membrane-based mechanoporation can be a cost-effective and efficient approach to pre-treat the hard-to-deliver immune cells before electroporation, elevating the treatment homogeneity and delivery of exogenous cargoes to a higher level.

Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.

Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.

Non-coding RNAs underlying chemoresistance in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a major health issue in the Western world. Current clinical imperatives for this disease include the identification of more effective biomarkers to detect GC at early stages and enhance the prevention and treatment of metastatic and chemoresistant GC. The advent of non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long-non coding RNAs (lncRNAs), has led to a better understanding of the mechanisms by which GC cells acquire features of therapy resistance. ncRNAs play critical roles in normal physiology, but their dysregulation has been detected in a variety of cancers, including GC. A subset of ncRNAs is GC-specific, implying their potential application as biomarkers and/or therapeutic targets. Hence, evaluating the specific functions of ncRNAs will help to expand novel treatment options for GC. CONCLUSIONS: In this review, we summarize some of the well-known ncRNAs that play a role in the development and progression of GC. We also review the application of such ncRNAs in clinical diagnostics and trials as potential biomarkers. Obviously, a deeper understanding of the biology and function of ncRNAs underlying chemoresistance can broaden horizons toward the development of personalized therapy against GC.

MAML1 promotes ESCC aggressiveness through upregulation of EMT marker TWIST1.

BACKGROUND: Mastermind-like 1 (MAML1) is the main transcriptional co-activator of Notch signaling pathway. It plays essential roles in several pathways including MEF2C, p53, Nf-кB and Wnt/ß-catenin. TWIST1 is known as a regulator of epithelial mesenchymal transition (EMT), which is considered as a primary step in promotion of tumor cell metastasis. Since concomitant expression of these genes was observed in tumors, our aim in this study was to elucidate the linkage between MAML1 and TWIST1 co-overexpression in esophageal squamous cell carcinoma (ESCC). RESULTS: While MAML1 silencing significantly down-regulated TWIST1, its ectopic expression up-regulated TWIST1 expression in both mRNA and protein levels in KYSE-30 cells. Expression of mesenchymal markers was increased significantly after MAML1 and TWIST1 ectopic expression, while epithelial markers expression was significantly decreased after silencing of both genes. Concomitant protein expression of MAML1 and TWIST1 was significantly observed in ESCC patients. Enforced expression of TWIST1 had no impact on MAML1 gene expression in KYSE-30 cells. CONCLUSION: The results clearly suggest transcriptional regulation of TWIST1 by MAML1 transcription factor in ESCC cells KYSE-30. Since TWIST1 is known as an EMT inducing marker, our results may revealed the mastermind behind TWIST1 function and introduced MAML1 as an upstream master regulator of TWIST1 and EMT in KYSE-30 cells.