Highly efficient genome editing via CRISPR-Cas9 ribonucleoprotein (RNP) delivery in mesenchymal stem cells.

The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Due to their desirable properties, mesenchymal stem cells (MSCs) have recently emerged as highly promising therapeutic agents, which properties include differentiation ability and cytokine production. While CRISPR-Cas9 technology is applied to develop MSC-based therapeutics, MSCs exhibit inefficient genome editing, and susceptibility to plasmid DNA. In this study, we compared and optimized plasmid DNA and RNP approaches for efficient genome engineering in MSCs. The RNP-mediated approach enabled genome editing with high indel frequency and low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfully generated B2M-knockout MSCs, which reduced T-cell differentiation, and improved MSC survival. Furthermore, this approach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs offer potential as a promising therapeutic strategy. [BMB Reports 2024; 57(1): 60-65].

Neuropathic Pain Component in Patients with Cervical Radicular Pain: A Single-Center Retrospective Study.

Background and Objectives: Evidence regarding the prevalence of neuropathic pain in patients with cervical radicular pain is limited. This study aimed to investigate the prevalence of neuropathic pain components in patients with cervical radicular pain using established screening tools and identify the relationship between neuropathic pain components and clinical factors. Materials and Methods: Data from 103 patients (aged ≥ 20 years) with cervical radicular pain who visited our pain clinic were analyzed retrospectively. Demographic characteristics, history of neck surgery, pain intensity using numeric rating score, dominant pain site, duration of symptoms, and neck disability index were assessed. The prevalence of neuropathic pain components was defined according to the Douleur Neuropathique 4 questions and painDETECT questionnaire tools. Patient characteristics were compared using the chi-square test or Fisher's exact test for categorical variables and the independent t-test or Mann−Whitney U test for continuous variables. The correlation between neck disability index and other variables was analyzed using Pearson's correlation coefficient. Results: Of the 103 patients, 29 (28.1%) had neuropathic pain components. The neck disability index was significantly higher (p < 0.001) for patients in the neuropathic pain group (23.79 ± 6.35) than that in the non- neuropathic pain group (18.43 ± 7.68). The Douleur Neuropathique 4 questions (r = 0.221, p < 0.025) and painDETECT questionnaire (r = 0.368, p < 0.001) scores positively correlated with the neck disability index score. Conclusions: The prevalence of neuropathic pain components in patients with cervical radicular pain was low. The patients in our study showed a strong correlation between functional deterioration and their neuropathic pain screening score. This study may be useful in understanding the characteristics of cervical radicular pain.

Selection and characterization of peptides specifically binding to HIV-1 psi (psi) RNA.

The packaging of HIV genomic RNA is mediated by a specific interaction between a nucleocapsid (NC) protein and packaging signal (psi, psi) RNA sequence. However, this interaction can be inhibited by the presence of peptides or proteins that specifically bind to the psi sequence. The 125-base-long psi RNA comprises a specific secondary structure that can be recognized by certain peptide sequences. Accordingly, the current study presents a method for selecting such peptides from a phage-displayed peptide library and characterization of resulting peptides in vitro. The RNA was covalently immobilized in a Covalink module using a carbodiimide condensation reaction at its 5'-end, leaving the proper secondary structure exposed and readily accessible. A phage display random peptide library was then screened against the RNA structure, and after five rounds of biopanning, enriched peptide sequences and conserved amino acid frames appeared. One of the enriched peptides was tested and shown to bind to psi RNA in a dose-dependent manner, plus it competed effectively with the NC protein as regards binding with the target RNA.