Identify favorable immunological profiles suitable for personalized treatments with RNA nanoparticles
(Spotlight on Nanowerk) Therapeutic nucleic acids (TNAs) and nucleic acid nanoparticles (NANPs) are designed to trigger specific intracellular responses beneficial for various biomedical applications. TNAs are becoming particularly attractive to researchers and clinicians because their functional versatility, programmability, and modularity hold great promise for the treatment of disorders such as viral infections, cancers, and genetic disorders.
However, the adverse stimulation of the innate immune system by TNAs has proven to be a major impediment to the accelerated transition and wider applications of TNAs in the clinical setting. The ability to define how TNAs interact with the immune system is highly desirable and becomes possible through the incorporation of TNAs onto specifically structured NANPs which can also support the synchronized delivery of therapeutic cocktails composed of different TNAs.
Using NANPs as scaffolds allows TNAs to be presented in an orderly fashion, which in turn provides flexibility in delivering selected TNAs for personalized treatments.
Previous work has shown that immune responses to NANPs change with various factors such as dimensionality (3D vs 2D), composition (DNA vs RNA), and functionalization with TNAs.
A work in progress published in Advanced functional materials (“Expanding Structural Space for Immunomodulatory Nucleic Acid Nanoparticles (Nanps) via Spatial Arrangement of Their Therapeutic Moeties”) by a team of scientists from the University of North Carolina at Charlotte, Nanotechnology Characterization Lab and Clemson University, investigated further the effect of functionalization on the immunorecognition of NANPs.
For this, a panel of NANPs composed entirely of RNA functionalized with Dicer Substrate (DS) RNAs positioned in all possible orientations has been extensively characterized in various reporter cell lines and animal models. Functional DS RNAs are TNAs designed for intracellular cutting that release siRNAs capable of silencing the production of specific target genes. The representative NANPs in the panel were chosen to be programmable hexagonal rings composed of six monomers capable of carrying up to six TNAs reliably placed in any position.
The research team used various methods to study the structural parameters and intracellular activity of the panel, including uptake in various mammalian cells, evaluation of their gene silencing efficiency, and immune stimulation of reporter cell lines. , all of which were followed by the study of immunostimulation in animals. models. Finally, trends across the panel were further investigated using peripheral blood mononuclear cells (PBMCs) from human donors to assess inter-person variability and the suitability of this approach for the personalized medicine.
Using immune reporter assays, the team examined the activation of various pathways (eg, IRF and NF-kB) to determine which cytokines are produced in response to each NANP treatment. These assays showed significant immunostimulation in response to all NANPs bearing DS RNAs. However, the unfunctionalized rings did not lead to detectable stimulation.
Interestingly, activation of retinoic acid-inducible gene-I (RIG-I), one of the key cytosolic immune receptors for detection of foreign RNA, was found to be dependent on the arrangement of DS RNAs around the NANP, with the higher number of DS RNAs hampering the RIG-I response compared to those with spacing between the TNAs.
The live Cytokine analysis results further confirmed that TNA orientation plays a role in immune activation. Another exciting finding from these experiments was that there was some cytokine production in the brain, indicating that NANPs could potentially be delivered across the blood-brain barrier using poly(lactide-co-glycolide)-graft -polyethyleneimine as carrier. This indicates that delivery of therapeutic NANPs to the central nervous system may be an achievable goal in the near future.
Overall, the extent of functionalization of NANPs with TNAs and their relative orientations showed a distinct pattern of immunostimulation in both human PBMCs and animal models. Rings that were functionalized with the more widely spaced TNAs led to higher levels of immune response in reporter cell lines and PBMC cultures.
This finding indicates that the extra space may allow for more efficient receptor binding, which in turn leads to the observed differences in immunostimulation. This differential response shows that the tertiary structure of NANPs can influence their delivery and interactions with cognate PRRs, resulting in varying levels of immune stimulation.
By understanding the immunorecognition of NANPs and tailoring the design and functionalization of NANPs based on the level of immune response desired, the scaffold itself can be tuned from an immunoquiescent platform to an immunostimulatory platform by incorporating TNAs in specific positions.
Advancing the current TNA approach to personalized medicine offers new strategies to address relevant public health challenges while providing a biodegradable and functional platform with tunable immune responses. The modularity of this platform allows it to function as a custom drug delivery platform, and treatment options can be customized based on desired applications for optimal drug design.
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