ABSTRACT
MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.
ABSTRACT
Early complications post hematopoietic stem cell transplantation (HSCT) such as sinusoidal obstruction syndrome (SOS) and graft versus host disease (GVHD) can be life threatening. Although several biomarkers have been identified to correlate with these complications and their response to treatment, these are yet to be used in clinical practice. Here, we evaluated circulating endothelial cells (CECs) (n = 26) and plasma biomarkers (ST2, REG3α, VCAM1, ICAM1, TIM3) (N = 210) at early time points, to determine their association with early complications post-HSCT. Elevated CEC counts at the end of conditioning was associated with GVHD, indicating endothelial damage during HSCT. Plasma levels of REG3α, VCAM1, ICAM1, and TIM3 on day 14 (D14) and D14 ICAM1 and D28 ST2 were significantly higher in patients with SOS and aGVHD, respectively. Upon sub-group analysis, D28 ST2, D14/D28 REG3α, and D14ICAM1 levels were significantly higher in patients with gastrointestinal GVHD, while D28ST2 was higher in those with skin/liver GVHD. High ST2 levels on D28 was significantly associated with non-relapse mortality (NRM) and overall survival. Our results suggest that elevated ST2 levels on D28 could predict the likelihood of developing aGVHD and could influence NRM and OS.
ABSTRACT
Therapeutic options for acute myeloid leukemia (AML) have remained unchanged for nearly the past 5 decades, with cytarabine and anthracyclines and use of hypomethylating agents for less intensive therapy. Implementation of large-scale genomic studies in the past decade has unraveled the genetic landscape and molecular etiology of AML. The approval of several novel drugs for targeted therapy, including midostaurin, enasidenib, ivosidenib, gemtuzumab-ozogamicin, and CPX351 by the US Food and Drug Administration has widened the treatment options for clinicians treating AML. This review focuses on some of these novel therapies and other promising agents under development, along with key clinical trial findings in AML.
