Prime Editing and DNA Repair System: Balancing Efficiency with Safety.

Prime editing (PE), a recent progression in CRISPR-based technologies, holds promise for precise genome editing without the risks associated with double-strand breaks. It can introduce a wide range of changes, including single-nucleotide variants, insertions, and small deletions. Despite these advancements, there is a need for further optimization to overcome certain limitations to increase efficiency. One such approach to enhance PE efficiency involves the inhibition of the DNA mismatch repair (MMR) system, specifically MLH1. The rationale behind this approach lies in the MMR system's role in correcting mismatched nucleotides during DNA replication. Inhibiting this repair pathway creates a window of opportunity for the PE machinery to incorporate the desired edits before permanent DNA repair actions. However, as the MMR system plays a crucial role in various cellular processes, it is important to consider the potential risks associated with manipulating this system. The new versions of PE with enhanced efficiency while blocking MLH1 are called PE4 and PE5. Here, we explore the potential risks associated with manipulating the MMR system. We pay special attention to the possible implications for human health, particularly the development of cancer.

IL-13Rα2 Status Predicts GB-13 (IL13.E13K-PE4E) Efficacy in High-Grade Glioma.

High-grade gliomas (HGG) are devastating diseases in children and adults. In the pediatric population, diffuse midline gliomas (DMG) harboring H3K27 alterations are the most aggressive primary malignant brain tumors. With no effective therapies available, children typically succumb to disease within one year of diagnosis. In adults, glioblastoma (GBM) remains largely intractable, with a median survival of approximately 14 months despite standard clinical care of radiation and temozolomide. Therefore, effective therapies for these tumors remain one of the most urgent and unmet needs in modern medicine. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface transmembrane protein upregulated in many HGGs, including DMG and adult GBM, posing a potentially promising therapeutic target for these tumors. In this study, we investigated the pharmacological effects of GB-13 (also known as IL13.E13K-PE4E), a novel peptide-toxin conjugate that contains a targeting moiety designed to bind IL-13Rα2 with high specificity and a point-mutant cytotoxic domain derived from Pseudomonas exotoxin A. Glioma cell lines demonstrated a spectrum of IL-13Rα2 expression at both the transcript and protein level. Anti-tumor effects of GB-13 strongly correlated with IL-13Rα2 expression and were reflected in apoptosis induction and decreased cell proliferation in vitro. Direct intratumoral administration of GB-13 via convection-enhanced delivery (CED) significantly decreased tumor burden and resulted in prolonged survival in IL-13Rα2-upregulated orthotopic xenograft models of HGG. In summary, administration of GB-13 demonstrated a promising pharmacological response in HGG models both in vitro and in vivo in a manner strongly associated with IL-13Rα2 expression, underscoring the potential of this IL-13Rα2-targeted therapy in a subset of HGG with increased IL-13Rα2 levels.

Exploration of some new secretory proteins to be employed for companion diagnosis of Mycobacterium tuberculosis.

Tuberculosis (TB) is a highly infectious disease and its early and precise diagnosis is essential to reduce morbidity and mortality of patients. Since the routine diagnostic tests (like Monteux, AFB smear microscopy, chest X-Ray) do not give infallible results, additional tests are always recommended. Therefore to address the concerns about non-specificity of the present battery of diagnostic tests, we have attempted to analyze some unique secretory antigens which could be able to identify the stage specific infection of MTB. In this study, we have used recombinant proteins CFP-10, ESAT-6, Ag85 A, Ag85B, Ag85C, PE3, PE4 and Mycp1 to eliminate heterogeneity and cross reactivity in clinical diagnosis. Amplified genes were cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinantly purified proteins were used as antigens against 158 sera samples of TB patients. Secretory proteins showed better response than the PPD control. Among all the used antigens PE3 and PE4 proteins showed better reactivity levels among all the groups of TB patients. The secretions of CFP-10 and ESAT-6 were also higher as compared to other secretory proteins like Ag85 A, Ag85B, Ag85C and MycP1.The clinical use of these newly identified secretory antigens could be of significant value for the confirmatory, rapid, simple and low-cost diagnosis of TB patients.