Main Conference - Day 2 (May 21)Wednesday, 21 May 2025

Oligonucleotides have advanced as potent tools in a number of applications including therapeutics and diagnostics. Alternative synthetic methods for the efficient, sustainable production of oligonucleotides are in dire need. In this presentation, I will highlight recent research from my lab involving modified nucleotides and oligonucleotides combined with polymerases and ligases.

Efficient enzymatic and microbial technologies are transforming the production of nucleic acid-based drugs, vaccines and biologics. Improved RNA polymerases enable cost-effective synthesis of high-quality mRNA with diverse sequences and modifications. Enzymatic oligonucleotide synthesis offers rapid, efficient and eco-friendly production of therapeutic RNA and DNA. Robust microbial protein production hosts meet the rising demand for high-quality enzymes used in nucleic acid synthesis, as well as therapeutic proteins and vaccine antigens.

As enzymatic oligonucleotide manufacturing processes gain traction among major siRNA therapeutics developers and CDMOs, the analytical control strategy for various enzymatic oligonucleotide synthesis pathways becomes the focal point when considering the adoption of ligation-based or fully enzymatic approaches. This presentation will showcase the impurity profile, product yield, and output reproducibility of different enzymatic processes and compare these approaches to phosphoramidite chemistry-based oligo synthesis. We will also discuss analytical and regulatory CMC considerations for enzymatic synthesis.

Small molecules and peptides by exhibiting rapid distribution into tumors and swift clearance from circulation offer an advantage over larger biologic molecules in delivering radiation to tumors safely. However, these two modalities often suffer from insufficiently long tumor retention, resulting in suboptimal efficacy. We are reporting here the use of covalency as a new strategy for the prolongation of tumor retention in the case of FAP-directing theranostics. Data with additional proprietary approaches will be also discussed.

Somatostatin receptor 2 (SSTR2)-targeted radioligand therapy is being expanded from the currently approved neuroendocrine tumors to small cell lung cancer and other SSTR2-postive tumors. The most advanced alpha-therapy is ²²⁵Ac-DOTATATE currently at Phase III. Here we will disclose the preclinical data of ⁶⁸Ga/²²⁵Ac-FL-031, a novel next generation theranostic pair.

EnPlusOne is enabling the future of RNA therapeutics. Its ezRNA™ platform is a next in class innovation that harnesses the power of enzymes to synthesize better RNA. RNA therapeutics have emerged as a commercially successful modality with a robust clinical pipeline that is trending from rare disease to prevalent indications. These therapeutics hold the promise of delivering improved medicines, however, drawbacks with current manufacturing approaches will continue to cause significant headwinds that threaten this new class from reaching its full potential. EnPlusOne is building the technology to solve this. It’s enzymatic, template-free, water-based approach promises to unlock sustainable and scalable commercial manufacturing of RNA therapeutics. The platform has demonstrated siRNA synthesis lengths (23-nt) and modifications (2’-F, 2’-OMe, and phosphorothioate backbone) culminating recently in the total synthesis of the Leqvio® antisense strand, a commercially approved siRNA treatment for high-cholesterol. The platform is directed in three critical areas: (1) solving the siRNA scale problem; (2) solving the gRNA purity problem; and (3) accessing enabling modifications for better RNA design. The presentation will cover the core technology platform, current synthesis performance, scaling efforts, and a cost and facility comparison between ezRNA™ and phosphoroamidite technologies at scale.

DNA and RNA microarrays are collections of nucleic acid molecules attached to a surface. We discuss advances in the chemistry that allows for the in situ synthesis of nucleic acid arrays of any kind, and present recent results in the production of sequence libraries.

Direct chemical synthesis of oligos with up to 1,728 nucleotides will be presented. The long oligos are characterized by Sanger sequencing. For the 1,728-mer, oligo yield is 0.016% or 41 pmol per gram of support. The success rate defined by percentage of correct sequences is 44%. Progress of long RNA synthesis will also be presented.

In the past decade, the interest in combining therapies and diagnostics has boomed and turned into a field of its own: theranostics. Major cancer centers across the United States, Europe and around the world have all opened dedicated theranostics centers. The approach requires a ligand that will specifically bind to the tumor, usually linked to a radioactive isotope for diagnosis by PET-scan or SPECT-imaging. Then, by using the same ligand and linker molecule connected to a different radionuclide, physicians are also able to treat the tumor. In this lecture, we present the discovery and pre-clinical development of [²¹²Pb]-PSV359, a macrocyclic CLIPS^TM peptide with high binding affinity and specificity against the Fibroblast Activation Protein Alpha (FAPa). In addition to this, we present first-in-human data for this lead that is currently in clinical development. FAPa is a 170 kDa homodimeric serine protease that is overexpressed in activated stromal fibroblasts of more than 90% of all human carcinomas [1,2]. Stromal fibroblasts play an important role in the development, growth and metastasis of cancer cells. However, FAPa expression under physiological conditions is very low in the majority of adult tissues.

Peptide synthesis has traditionally relied on Classic Liquid-Phase Peptide Synthesis (LPPS) and Solid-Phase Peptide Synthesis (SPPS), each with inherent limitations. LPPS, while efficient in reagent use, is only suitable for short peptides and requires tedious isolation steps. SPPS, widely adopted, is solvent-intensive and presents challenges in large-scale manufacturing. TAG-Assisted Peptide Synthesis (TAPS) combines the best of both worlds, offering an innovative hybrid approach that integrates LPPS and SPPS advantages. This method employs a TAG-anchor molecule, which solubilizes the growing peptide in an organic solution, allowing for multiple elongation cycles without isolation, significantly improving efficiency. TAPS presents a highly efficient, sustainable, and scalable alternative to traditional peptide synthesis. By leveraging solution-phase chemistry, online monitoring, and process automation, it enables improved manufacturing flexibility, cost reduction, and environmental sustainability. With patent protection and proven feasibility on GLP-1 peptides, TAPS is set to redefine peptide production for pharmaceutical applications.

Agilent has developed new approaches for purification of single strand and duplex nucleic acids. This talk will cover techniques for polish purification of duplex RNA with diafiltration and a dual mode chromatography approach for challenging RNA sequences.

Anion exchange chromatography is crucial in oligonucleotide manufacturing. Traditional scale-up methods often rely on fixed bed heights and linear velocities, but recent trends for biologics favor volume-based scale-up to improve operational efficiency and process robustness at large scales. This presentation showcases a case study of anion exchange purification for phosphorodiamidate morpholino oligomers (PMOs), highlighting the advantages of a volume-based approach in enhancing throughput, reproducibility, and scalability. These findings underscore the benefits of volume-based scale-up as a viable strategy to meet the growing demand for oligonucleotides.

Through CIPPS, we explored a large and diverse panel of peptides to understand how they permeate cells and cross membranes. I will discuss insights into the mechanisms by which peptides enter cells, the role of peptide-lipid interactions in these processes, and strategies to reengineer membrane-active peptides for targeting intracellular molecules.

The genetically-encoded Microcycle screening platform enables the rapid intracellular generation and high-throughput screening of a cyclic hexa-peptide library of 3.2 million members within disease-relevant mammalian cell lines. Examples will be presented that illustrate the inherent advantages of intracellular targeted screens (over traditional in vitro approaches), and the scaffold hopping of a pharmacophore into a small molecule backbone (enabled by the conformational rigidity of cyclic hexa-peptides).

Cyclotides are ultrastable macrocyclic peptide scaffolds derived from plants. They can be readily synthesized using solid phase peptide synthesis and used to deliver bioactive peptide epitopes to a variety of extracellular or intracellular targets. This presentation will describe how the biophysical properties of cyclotides facilitate their intracellular targeting and their applications to block protein:protein targets implicated in cancer.

RNA therapeutics have great potential to selectively treat human disease, especially cancer, COVID and neurological disorders. However, due to their 8-14 kDa size and 20-40 negatively charged phosphates, RNAs therapeutics have exceedingly limited ability to overcome a billion years of evolutionary defenses that prevent them from escaping across the endosomal lipid bilayer membrane into the cytosol of cells. Indeed, <1% of siRNAs, ASOs, PMOs actually escape the endosome. Consequently, endosomal escape remains The Technological Problem to solve before we can begin development of RNA therapeutics to tackle the myriad of currently undruggable diseases.

Nitto BioPharma has developed a tool-box of novel lipids and formulations to enable efficient delivery of mRNA to cells. We highlight the ability to efficiently deliver an mRNA to lung, liver, and T cells in vivo, leveraging our broad repertoire of biodegradable lipids to establish safe and robust cellular delivery.

The development of robust, scalable processes for manufacturing oligonucleotides is critical to advance therapeutic applications, including gene editing, RNA therapies, and antisense oligonucleotides. As oligonucleotide therapeutics emerge as transformative treatments for diseases like genetic disorders and cancers, successful commercialization relies heavily on improvements in manufacturing techniques. This talk will explore the challenges and strategies in oligonucleotide process development, focusing on optimizing solid-phase synthesis and purification steps to enhance production efficiency. Key topics will include innovative approaches for overcoming manufacturing challenges, cutting-edge analytical techniques to ensure product consistency and methods to accelerate time to market. Special emphasis will be placed on controlling impurities, ensuring high yields, and addressing issues related to solubility and sequence complexity. The session will also highlight future directions for improving scalability and regulatory approaches in oligonucleotide manufacturing, with a particular focus on the role of Nitto Avecia in supporting the growing pipeline of oligonucleotide-based therapies and facilitating their broader clinical application.

The emergence and spread of drug resistance challenges our ability to control infectious diseases. In response, we are developing peptide-based drugs based on a cyclic peptide engineered from a human defense protein. Using late-stage conjugation strategies, we produced stable and selective peptide-drug conjugates with cargo driven potencies toward intracellular targets.

Rising awareness of dietary proteins and bioactive peptides (BPs) has driven research and commercial interest. BPs, sourced from food proteins, offer potential as functional foods and nutraceuticals with various health benefits. This presentation will explore the discovery-to-validation process for plant, fungi, and algae-derived bioactive compounds.