ADARs are a new class of oligo-directed precision medicines to address a myriad of previously undruggable targets. At Korro, we use ASOs to recruit endogenous ADARs, to carry out (A-to-I) edits that can repair G-to-A mutations and modulate protein function by changing the amino acid code. This presentation will focus on Korro’s approach to ADAR-mediated RNA editing and provide an update on progress towards clinic.

Wave’s AIMers are oligonucleotides that engage endogenous ADAR enzymes to induce highly efficient and specific A-to-I RNA base editing. CTA submissions are expected in 2H 2023 for our first RNA editing program, WVE-006, which aims to correct a disease-causing protein-coding mutation in a liver mRNA. We provide an update on our preclinical efforts to broaden the potential applications for RNA editing by expanding the target space and tissue targeting capabilities.

We developed self-amplifying RNA and modified RNA platforms into vectors capable of carrying synthetic circuitry payloads that can provide a variety of desirable dynamics. We also encoded miRNA target sites on our RNA vectors to provide for highly specific cell type classification. We are using this technology to create next-generation cancer immunotherapy RNA vectors capable of activating therapeutic payloads discriminately in cancer cells.

mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides, N1-methylpseudouridine, to reduce cellular immunogenicity and increase protein production. We have developed a method, direct analysis of ribosome targeting, DART, to screen modified mRNAs for increased translation by human ribosomes. Our results identify small changes in 5′ UTR sequence and chemical modification that increase protein production in human cells.

mRNA translation is tightly regulated in mammalian cells. In this talk, I will present high-resolution 3D in situ sequencing approaches (STARmap, RIBOmap, TEMPOmap) that enable simultaneous mapping of RNA lifecycle of thousands of genes within intact cells and tissues. Following that, I will explore strategies to improve mRNA translation and stability using mRNA-oligonucleotide conjugates.

Modified mRNA technology has been successfully applied in the development of vaccines, most recently against SARS-CoV2 virus and novel variants in the fight against COVID-19. In oncology, while several novel immunotherapies have demonstrated striking responses in several hard-to-treat cancers, there is still a need for innovative therapeutic approaches to overcome mechanisms of relapse, resistance and immune suppression. I will present some of our efforts to develop novel mRNA-based therapies that activate the ability of the immune system to develop durable anti-tumor immunity.

Linear messenger RNA (mRNA) is being investigated for other therapeutic applications. We are developing a new class of synthetic, protein-coding, circular RNA (oRNA). It is a scalable, cap-independent, and immunoquiescent protein-coding RNA platform. Strategies and results of optimizing oRNA to drive high protein expression and durability will be presented. When combined with an efficient immune cell delivering LNP, effective in situ CAR T tumor killing is observed in preclinical models.

Irrespective of the specific drug delivery system employed, the inherent druggability of mRNA molecules can be significantly enhanced through the optimization of either translatability or thermal stability. In a recent endeavor, we developed a novel algorithm—LinearDesign, derived from natural language processing—to identify optimal vaccine molecules characterized by greatly augmented immunogenicity. This innovative approach extends its applicability beyond conventional linear mRNA molecules to encompass circular variants as well.

Here we develop and deploy CodonBERT, an mRNA codon optimization tool to optimize protein expression from delivered RNA. We also demonstrate Saluki, our state-of-the-art, hybrid convolutional and recurrent deep neural network which relies only upon an mRNA sequence annotated with coding frame and splice sites to predict half-life. Collectively, this group of tools represents the power of machine learning–guided approaches in the design of mRNA vaccines.

We describe a safe and efficient lipid nanoparticle (LNP) for the delivery of mRNA and sgRNAs that use novel amino-ionizable lipids and a strategy to target RNA payloads to specific cell types in vivo. We show selective and highly efficient in vivo therapeutic genome editing in glioma, ovarian, and blood cancers, with more than 80% editing and increased survival of 90%. This approach opens new avenues in cancer therapeutics.

In this talk, I will discuss our efforts towards the development of lipid nanoparticle (LNP) platforms that enable the delivery of RNA therapeutics and vaccines to a range of target cells and tissues in the body. Furthermore, I will describe new therapeutic strategies utilizing these LNPs including (i) in vivo reprogramming of immune cells for cancer immunotherapy and vaccination, (ii) in utero gene editing for treating disease before birth, and (iii) mRNA prenatal therapeutics for treating pregnancy disorders such as pre-eclampsia.

Circular RNAs (circRNAs) are a promising platform for mRNA-based therapeutics. However, the immunogenicity of circRNAs has not been studied using human immune cells. We found that while purified circRNAs are nonimmunogenic in A549 cells, they elicited strong immune responses in PBMC and macrophages. Using CRISPR knockout cell lines, we found that TLR7/8 mediates the innate immune sensing of unmodified circRNAs.

Eleven Therapeutics ushers in the next generation of RNA therapeutics by mastering combinatorial chemistry, synthetic biology, and AI. Our extended-release mRNA, dubbed xRNA, utilizes non-canonical building blocks to boost the durability of mRNA technologies. Our in vitro and in vivo experiments show superiority of this technology over standard mRNA, and our massively parallel assay is designed to deep-learn the SAR of xRNAs. Eleven’s pipeline addresses unmet medical needs in the metabolic, hematologic, and infectious disease areas, as well as prophylactic siRNAs for pandemic-poised viral infections. Eleven develops a complementary platform, DELiveri, utilizing proprietary Molecular Radar technology to screen ex-hepatic cell-penetrating delivery carriers.

The presentation will describe the generation and the use of protein-based nano-capsules to deliver mRNA across the blood-brain barrier to treat CNS diseases. The therapeutic potential of this delivery technology will be illustrated for the mRNA treatment of monogenetic CNS disorders such as metachromatic leukodystrophy (MLD), a lysosomal storage disease.