Researchers established a breakthrough gene and cell therapy system called human nitric oxide-responsive transgene regulation modality (hNORM) through the development of a regulation system that functions along with nitroglycerin (NG). The vasodilative compound NG functions as both a bioavailable and secure method for controlling pharmaceutical production directly within the body. The system provides precise and dynamically controlled reversible gene expression through transdermal delivery based on the novel transgene regulation method.
Through mitochondrial aldehyde dehydrogenase 2 (ALDH2), the system converts NG into nitric oxide (NO), which activates gene expression through cyclic guanosine monophosphate (cGMP)-dependent pathways. Preclinical research showed hNORM effectively controlled the production of therapeutic proteins used to treat type II diabetes and obesity. An innovation delivers personalized medicine through an alternative approach that permits easy and minimally invasive care for managing persistent diseases.
Researchers developed the hNORM system for mammalian cell-based NO detection by using HEK-293 cells as the primary platform. The trials revealed that NO donor compounds DETA, NONOate, and S-nitroso-N-acetylpenicillamine (SNAP) were unable to stimulate secreted alkaline phosphatase (SEAP) expression during a 24-hour observation period because of inadequate sensitivity. The addition of protein kinase G1(PKG1) during transfection procedures led to better signal amplification of cGMP-induced NO effects that ultimately activated SEAP expression. The research outcomes demonstrated why PKG1 represents a crucial component for boosting the delivery of cGMP signals, thereby enabling efficient NO detection.
The hNORM system worked effectively with diverse human and rodent cell lines from A549 to HeLa, HepG2, Chinese hamster ovary cells (CHO-K1), and HT-1080 and generated its best detection outcome with HEK-293 cells. A monoclonal HEKhNORM1 cell line with enhanced stability along with improved in vivo features was generated through the sleeping beauty transposase system, which used nanoluciferase (NLuc) as the powerful reporter. The pharmacological utility of methylene blue served as a safety mechanism to control transgene expression levels. The research establishes hNORM as a viable method for regulating genes through NO dependence in mammalian cellular environments.
The approval of recombinant human insulin over four decades ago sparked research interest in cell-based therapies because of ongoing problems with biopharmaceuticals, such as their manufacturing complexities and frequent administrations. Chimeric antigen receptor T (CAR-T) cell therapy- therapy demonstrates this transformation while requiring improved control methods to optimize its performance.
Transdermal delivery through topical patches presents a promising method to control implanted designer cells with enhanced specificity while improving patient compliance. The NG patch system functions as an external, non-surgical tool to control gene activation and differs from conventional, expensive injectable medicine administration systems. More research must be conducted to improve both long-term stability and clinical implementation potential as hNORM delivers affordable solutions for reducing medication dependence and improving patient compliance.
Experimental preparations used drugs and chemicals obtained from Enzo Life Sciences as well as Cayman Chemical and Sigma-Aldrich. The preparation of NO donors took place shortly before each lab session because these compounds show brief stability when dissolved in water. The research obtained NG transdermal patches at various dosages from Cito Pharma Services. The process of plasmid assembly used either restriction enzyme cloning with T4 Deoxyribonucleic acid (DNA) ligase or Gibson enzyme-based assembly methods. Transformed plasmids received XL10-Gold E. coli treatment, and the researchers obtained them through Zymo Research Kit extraction processes.
The researchers obtained data from SEAP and NLuc quantifications by using plate readers to create stable cell lines through Sleeping Beauty transposase and fluorescence sorting. The researchers evaluated cell health and toxic substance effects through AlamarBlue and Cytofluorometric Quantification of Nucleic Acids (CyQUANT) testing.
Standard protocols guided the execution of Western blotting and quantitative polymerase chain reaction (qPCR) while ribonucleic acid sequence (RNAseq) analyses. After following Illumina’s protocols, researchers generated RNAseq libraries before carrying out Illumina platform sequencing.
The study presents an innovative NO-sensitive molecular regulation mechanism that enables medical professionals to manage reversible therapeutic protein generation through standard nitroglycerin bandages. The system works through human mitochondrial aldehyde dehydrogenase, which transforms nitroglycerin into nitric oxide that starts a signaling sequence to activate target genes.
Proof-of-concept research showed that mouse cells embedded with this system managed obesity-related type 2 diabetes in mice by releasing glucagon-like peptide-1 gradually while controlling blood glucose. The method demonstrates potential as a new patient-centered technique to specifically administer biopharmaceutical substances when needed, which demonstrates promise for managing ongoing illnesses.
References: Mahameed M, Xue S, Danuser B, et al. Nitroglycerin-responsive gene switch for the on-demand production of therapeutic proteins. Nat Biomed Eng. 2025. doi:10.1038/s41551-025-01350-7
