Circular RNAs are widely generated across eukaryotic genomes. In fact, for some genes, the abundance of the circular RNA exceeds that of the associated linear mRNA by >10-fold. We are developing methods to identify/characterize circular RNAs as well as understand how the spliceosome selects exons for backsplicing. By characterizing these mechanisms in detail and identifying the functions of mature circular RNAs, we aim to reveal novel therapeutic targets and modalities.

Circular RNAs (circRNAs) are a novel class of RNAs distinguished by their covalently-closed topology. There are several challenges to working with circular RNAs including their low abundance NA, their lack of a unique feature, and their sequence similarity to linear RNAs. In this talk, we will describe our approaches to synthesize and purify circRNAs that enable their functional investigations and development into effective therapies.

Orna has developed a highly efficient circularization process for generating large quantities of purified circular RNA (oRNA). We demonstrate, in the absence of modified nucleotides, that oRNA is immunoquiescent via in vitro and in vivo assessment. We present case studies using novel ionizable lipids for the generation of lipid nanoparticles to systemically deliver oRNA therapies for oncology (in situ CAR) and protein replacement.

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not traditionally thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. RNA-glycan conjugates, or glycoRNAs are present in multiple cell types and tissues. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with members of the Siglec receptor family. New chemical tools and insights into the molecular nature of glycoRNAs will be presented.

Antigen-encoding small circRNA vaccines are highly stable and produce concatemeric antigens, resulting in robust and long-lasting adaptive immunity. Relative to several modified mRNAs, circRNA vaccines elicited up to 10-fold antigen-specific T cells in mice with superior safety. circRNA vaccines are widely applicable for tumor and viral (neo)antigens to elicit CD8+/CD4+ T cell responses in young or immunosenescent-aged mice. Combining circRNA vaccines with immune checkpoint blockade (ICB) reduced tumor immunosuppression and eradicated multiple types of murine tumors, including ICB-resistant BrafV600E melanoma. Moreover, pulmonary circRNA vaccines protected mice from influenza challenge. Overall, small circRNA vaccines are promising for versatile applications.

Lipid nanoparticles for mRNA vaccines and therapeutics are coated with polyethylene glycol (PEG) to aid in delivery of the mRNA. However, it is widely reported that a high percentage of humans have pre-existing antibodies to PEG. To better understand how pre-existing or treatment boosted anti-PEG antibodies impact delivery of the mRNA, a suite of assays were developed to isotype the anti-PEG antibody response.

Moderna has launched a respiratory combination vaccine program, mRNA-1230, to target three of the most significant viruses causing respiratory disease in older adults: the SARS-CoV-2 virus, influenza virus, and respiratory syncytial virus (RSV). This case study describes clinical assays developed to characterize the immune responses to various strains of the three viruses with a focus on the qualification strategy and results.

The role of pharmacometrics or modeling and simulation in the development of siRNA therapeutics and mRNA vaccines will be presented. The session will include the following topics:

• Translational modeling approaches for siRNA and mRNA therapeutics
• Modeling and simulation approaches for Phase III dose selection of siRNA therapeutics and mRNA vaccines
• Dose selection for special cases-- dose modeling for mRNA cancer vaccine and pediatric vaccine dose selection?

• Assay platforms for PK/PD, BioD, and immunogenicity
• Challenges in reagent identification and qualification
• Life-cycle maintenance for stability
• Qualification and bridging for new labeling, processing, and manufacturing
• Planning ahead and best practices for critical reagent management

Chemotherapy leads to loss of cardiomyocytes and leads to heart failure. Therefore, there is a need for therapeutics that can protect cardiomyocytes from cardiotoxic agents.  Recently, modified mRNA (modRNA) has emerged as a promising technology for cardiac therapeutics. Using modRNA design, CRISPR technology, and positively charged lipid nanoparticles, we created a cardiomyocytes-specific modRNA translational system that translates exclusively in cardiomyocytes and protects them within minutes after intravenous (IV) injection.

LNPs represent the most advanced nonviral nanoparticle delivery systems that have been extensively investigated for nucleic acid delivery. Here I will discuss the design and development of combinatorial synthetic bioreducible and biodegradable lipid nanoparticles (LNPs) with distinct chemical structures and properties for in vitro and in vivo intracellular mRNA delivery. I will discuss the utilization of a library screening strategy to identify optimal LNPs for organ and cell targeted mRNA delivery and showcase the applications of the optimized LNPs in cell engineering and genome editing. 

A novel siRNA nanomedicine specific to connective tissue growth factor (CTGF), an important regulator of fibrosis in both hepatic and renal cells, is developed. Nanomedicine is further targeted to asialoglycoprotein receptors on hepatocytes and renal tubular epithelial cells by surface modification with galactosamine ligand, to enhance the cell uptake. On animals this innovative construct showed long circulation time and high accumulation in hepatic and renal tissues, making it a promising mRNA therapeutic for liver and kidney fibrosis.

We describe a high-throughput chemical approach for the synthesis and screening of lipid nanoparticles (LNPs) composed of 720 unique ionizable lipids, which identified key structural features for biodegradable ionizable lipids. The intratracheal administration of lead mRNA-LNP formulations efficiently delivers genes to cells in pulmonary epithelial tissues. The gene editing capabilities were validated using a Cre and CRISPR-Cas9 model, providing a new gateway to treat pulmonary genetic disorders.