Poster X20 - Determination of siRNA and mRNA concentration in lipid nanoparticle co-formulation

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Deepali Bisht (she/her/hers)

Graduate Research Assistant
University of Texas at El Paso
El Paso, Texas, United States

Co-Author(s)

Denice P. Rout

Graduate Research Assistant
University of Texas at El Paso
El Paso, Texas, United States

Primary Investigator(s)

Wilson Poon, PhD (he/him/his)

Assistant Professor
University of Texas at El Paso
El Paso, Texas, United States

Introduction: The advancement in nanoparticle engineering for delivering biomacromolecules and pharmacological drugs has remarkably boosted translational research. Among the engineered nanoparticles, lipid nanoparticles (LNPs) have emerged as the reliable platform for delivering genes, including messenger RNA (mRNA) and small interfering RNA (siRNA). New studies take advantage of co-encapsulating these two types of RNA simultaneously in a single LNP formulation to upregulate/downregulate the desired genes to facilitate a synergistic therapeutic effect. The absolute quantification of the total encapsulated RNA in LNPs is well implemented in the field. However, the quantification of the separate mRNA and siRNA in a co-formulation has not yet been sufficiently explored. As the existing approach quantifies the total encapsulated RNA and the size of the mRNA and siRNA vary significantly, using the common method to quantify RNA loading will not provide effective specific RNA quantification. Thus, we aim to synthesize mRNA/siRNA LNP co-formulations and quantify the encapsulation efficiency of loaded mRNA and siRNA in the co-formulations.

Materials and

Methods: For our study, we used four different LNP formulations: empty LNP, mRNA-encapsulated LNP, siRNA-encapsulated LNP, and one co-formulation of mRNA/siRNA encapsulated LNP. The RNA was quantified by RNA gel quantification method using the Bio-Rad ChemiDoc Image Lab software. LNPs (mRNA, siRNA and mRNA/siRNA) were treated with Tris EDTA (TE) buffer to obtain unencapsulated RNA present outside LNPs, and treated with Triton-X (TX) to obtain the total RNA, which consisted of encapsulated and unencapsulated RNA. The dilutions of RNA standard for mRNA and siRNA were separately loaded on the RNA gels and the RNA standard curve was plotted. The TE and TX-treated LNP samples were loaded on the RNA gel to acquire two bands of mRNA and siRNA, based on the size difference. The standard curves were used to quantify the mRNA and siRNA based on band intensities. The encapsulation efficiency was estimated as per conventional methods as (Total RNA – unencapsulated RNA) / (Total RNA) x 100%. The estimated concentrations of the mRNA and siRNA were compared with the existing RNA and small RNA estimation kits.

Results, Conclusions, and Discussions: Result
We synthesized empty, mRNA, siRNA, and mRNA/siRNA LNPs with 5:1 and 10:1 N/P ratios and the standard SM-102 LNP lipid component ratios. These LNPs have sizes ranging from 120 nm to 130 nm, PDI from 0.05 to 0.189, and zeta potential from -0.8 mV to -1.98 mV as measured by the Malvern Zetasizer Ultra. We performed LNP encapsulation efficiency assays for mRNA and siRNA LNPs using two different kits - Promega QuantiFluor RNA quantification kit and ABP iQuant microRNA kit. Interestingly, we observed that both kits can quantify the RNA irrespective of its size and give comparable results despite what is intended by the manufacturers, which shows non-specificity towards the type of RNA. This motivated us to work on the research gap of developing a method to quantify the mRNA and siRNA in a co-formulation using RNA gel imaging quantification. Our preliminary results using the RNA gel imaging quantification demonstrated that it is able to resolve encapsulated and unencapsulated mRNA and siRNA from LNP formulations based on RNA size. We are currently optimizing the image analysis workflow of the band intensities using the Image Lab software.
Conclusion
From our experiments, we estimated that the existing method of quantifying encapsulated RNA using kits in mRNA/siRNA coformulation quantifies the total RNA irrespective of the size and type. With our new approach, we are determined to quantify the mRNA/siRNA ratio for a better dosage:effect estimation and designing a range of co-formulations.
Impact
The co-formulation of mRNA and siRNA in LNPs has strong potential to impact many polygenic or complex genetic diseases, where it is required to upregulate/downregulate certain synergistic genes. Quantifying the mRNA and siRNA concentration in a co-formulation is necessary for pre-clinical optimization to help understand the effect of mRNA on siRNA encapsulation; as well as in the clinical translation of these formulations for scale-up manufacturing and dosimetric studies.. Our new quantification method would allow a more rigorous understanding of critical quality attributes (CQA) in LNP-based co-formulation pharmaceutics, ultimately benefiting treatment outcomes of patients with complex genetic diseases by enabling improved pharmaceutical development and translation.

Acknowledgements (Optional): I would like to acknowledge the University of Texas System STARs Program (E210292976) and the Trauma Research and Combat Casualty Care Collaborative (TRC4) (226420284A) for the expansion of the project.