RNA-Based Gene Therapy Advances: Splicing Control, Nanoparticle Delivery, MicroRNA Platform

Researchers have reported new advances in RNA-based gene therapy, including a clinically approved small molecule that precisely controls gene expression through RNA splicing modulation, protein-coated nanoparticles for safer gene delivery, and a microRNA-derived platform for multitargeted cancer therapy. The approaches address key limitations of existing gene therapies, ranging from the risk of permanent genomic changes to immune responses and the need for combinatorial cancer treatment.

In a study published in Nature Communications, researchers described a strategy for precise gene regulation via RNA splicing modulation, utilizing a clinically approved small molecule. The small molecule, already in clinical use for unrelated indications, can be repurposed to manipulate splicing outcomes by binding to specific components of the spliceosome complex. This binding shifts the splicing equilibrium, promoting the inclusion or exclusion of targeted exons. Unlike gene editing techniques that alter the DNA code, this RNA-centric approach allows reversible, adjustable, and more nuanced gene control without permanent genomic changes. Varying the concentration and exposure duration of the small molecule enabled graded responses in splicing patterns, translating to dose-dependent changes in protein production, confirmed across multiple gene targets and cell types. Genetic diseases caused by splicing defects, such as spinal muscular atrophy or certain forms of cystic fibrosis, stand to benefit from a modality that can restore normal splicing patterns, and cancers driven by aberrant splicing isoforms could be sensitized to treatment by selectively switching splice variants. Experiments using patient-derived cells demonstrated functional rescue of disease phenotypes following treatment, with restoration of normal protein function. Transcriptomic analyses revealed a high degree of specificity, with minimal unintended splicing changes beyond the intended gene targets, and chronic exposure studies indicated that cells maintain viability and normal function.

Separately, scientists at the University of Michigan have pioneered a method to deliver gene therapies using protein-coated nanoparticles. The nanoparticles use serum albumin as the coating material, substituting fat-based lipid nanoparticles commonly used in mRNA vaccines and gene therapies, which have been linked to inflammation and hepatic toxicity. The team successfully modified multiple human cell types—including liver cancer, kidney, and immune cells—cultured in vitro, by encapsulating DNA or messenger RNA encoding green fluorescent protein (GFP) inside the nanoparticles. The protein encapsulation shields the genetic cargo and enhances biocompatibility by potentially reducing inflammatory responses and liver damage. The nanoparticles are fabricated using electrohydrodynamic (EHD) jetting technology, in which a solution of protein and genetic material is subjected to a high-voltage electric field, and subsequently coated with polyethylenimine, a positively charged polymer that facilitates endosomal escape. Unlike viral vectors, these protein nanoparticles deliver genetic instructions without integrating the nucleic acids into the host genome, reducing the risk of insertional mutagenesis. Because the genetic effects are transient—mRNA persists for days while plasmid DNA expression may last several months—protocols may involve repeated dosing or booster administrations. The team envisions future deployment of CRISPR-Cas9 gene editing elements within these nanoparticles to achieve targeted and permanent genome modifications, potentially establishing single-administration cures. This platform emerges amidst concern that FDA-approved viral therapies administered systemically risk provoking infections and dangerous immune reactions, and that viral vectors can inadvertently induce secondary malignancies.

In research on glioblastoma, investigators demonstrated a gene therapy platform based on accessory byproducts derived from microRNA processing. The platform is capable of carrying multiple, functionally different elements, which become biologically active upon cleavage from the primary sequence where they are encoded. The only requirement for its engineering is the preservation of Drosha cleaving sites, which allows the proper release of each active component, offering a flexible, plug-and-play scaffold for disease-specific multitargeting. As a proof of principle, the approach was validated in various models of glioblastoma, the deadliest brain cancer in adults, translating into a highly synergistic antitumor effect. The work represents the first time that accessory sequences making up the scaffolding component of a microRNA gene are observed to persist following its processing, allowing the microRNA genetic structure to be revisited as a vessel for introducing a combination of diverse, polyfunctional small ncRNAs into cells. The design also allows cleavage of a properly sized aptamer structure from a longer RNA polymerase II–driven sequence, overcoming the previous limitation that aptamers depend on RNA polymerase III for intracellular expression; the investigators used an aptamer against p50 to demonstrate this strategy.