Transforming Drug Discovery With Oligonucleotide Therapeutics

Accelerating Oligonucleotide Therapeutics Comprehensive solutions from target ID to IND Interview with Dr. Monia, Ionis Pharmaceuticals p. 05 siRNA & ASO Design p. 14 Crucial Oligo Safety Considerations p. 20 1 The Spark of Innovation in Oligonucleotide Therapeutics 03 2 Evotec’s Letter from the Editor 04 3 Interview with Dr. Monia, CEO of Ionis Pharmaceuticals 05 4 End-to-End Oligonucleotide Platform 13 in-silico Oligonucleotide Design 14 Oligonucleotide Chemistry and Synthesis 15 Efficacy (Target Engagement in vitro) 18 ASO Screening and Profiling 18 in vitro Safety 20 Prediction of Hepatotoxicity (Caspase 3/7) 20 Prediction of Immunotoxicity (Bjab and hPBMCs) 21 Prediction of Neurotoxicity (MEA) 23 Off-target Analysis 25 PK/PD Characterization of Oligonucleotides 26 in vivo Studies 27 Bioanalytical Capabilities 28 – Quantitative methods 30 • LC-MS/MS 30 • SplintR Ligase qPCR 31 • QuantiGene™ 33 • ASO Immunodetection Techniques SMxPRO/MSD 34 • Semi-quantitative and Qualitative Methods 37 - High Content Quantitative Image Analysis 37 - High Content Imaging 38 in vitro ADME Characterization: in vitro Stability and Metabolic Profiling 39 in vitro ADME Characterization: Plasma Protein Binding 40 PK/PD Modeling 41 Pathology 44 Preclinical Development 46 Species Selection 46 Toxicological Profile 48 Preclinical Toxicology Study Design 49 Surgical Capabilities 49 Safety Pharmacology 50 Genotoxicology Profile 51 Regulatory Framework and Considerations 51 Quality and Compliance 52 Regulatory Support 53 Therapeutic Area Expertise 54 5 Closing remark 55 CONTENTS 3 I n the rapidly evolving landscape of precision medicine, oligonucleotide therapeutics are heralding a new era of targeted treatments. These groundbreaking molecules – capable of modulating gene expression with unmatched specificity – represent a paradigm shift in addressing genetic and rare diseases. From neuroscience to metabolic disorders, they offer hope for conditions once considered untreatable. Unlike traditional drugs, oligonucleotides target RNA, reaching the root of disease pathways. By their very design they minimize off-target effects and open up possibilities for transformative therapies such as RNA editing, protein upregulation and exon skipping. With over 20 approved drugs and many more in clinical trials, the field has shifted from high-risk newcomer to demonstrated clinical potential. Evotec stands at the forefront of this revolution, combining scientific excellence with the ability to take an integrated, end-toend approach to drug discovery and development. Through relentless innovation and collaboration, we have turned challenges into opportunities, empowering our customers to accelerate the journey from concept to clinic. This eBook invites you to explore Evotec’s oligonucleotide journey, cutting-edge technologies and our path towards transformative patient care through precision medicine. Dive deeper into the oligonucleotide therapeutics revolution in an interview with the CEO of Ionis Pharmaceuticals. Learn how RNA-targeted therapeutics, including antisense oligonucleotides (ASOs) and siRNAs, are innovating the pharmaceutical industry offering enhanced delivery and potency, while reducing off-target side effects. And gain expert guidance on how to develop groundbreaking oligonucleotide therapeutics, from concept through to the clinic. Find the latest insights on aspects including in silico design, chemistry and synthesis, efficacy, safety, bioanalysis, stability and metabolic profiling, preclinical development and more. The Spark of Innovation in Oligonucleotide Therapeutics 4 Do you have a target idea that you haven’t been able to find a drug for? A favorite kinase or something that has so many family members that targeting it with a small molecule would be problematic. Do you want to cure a rare disease? Maybe your target is a solution for a global epidemic like MASH or obesity? Oligonucleotide drugs have proven their worth as serious contenders for drug makers. At first this modality was too new and there were too many regulatory hurdles, oligo drugs were on the edge and only considered for monogenic diseases where no other treatment was available and what was considered a big risk, was worth taking. But with 20 marketed drugs and many 100s more authorized for clinical trial use this modality is catching up fast, the cost to establish a proof of concept for the druggability of a target and its suitability for this modality needs only the time it takes to design and screen a library for hits, less than 6 months. Trying to upregulate protein expression? Modify splicing or just knocking down target RNA with the right team you can get to candidate in 2 years. Maybe you just need help setting up a phenotypic assay? A complex disease model or novel mechanism of action, maybe just some DMPK or regulatory advice. In this eBook we detail how we go about our oligonucleotide projects from target ID to preclinical and clinical development. We hope you find the information valuable. If you would like to discuss with us personally, please feel free to reach out at info@evotec.com. Evotec’s Letter From the Editor Hilary Brooks VP and Modality Lead for Oligonucleotide Therapeutics 5 For his cutting-edge insights into oligonucleotide therapies, we interviewed Dr. Brett Monia, Ph.D., at Ionis Pharmaceuticals. The company is a leader in the field, with groundbreaking technology platforms to develop oligonucleotides, including ASOs and siRNAs and advanced chemistries and capabilities. In this interview, Brett talks us through his journey with Ionis Pharmaceuticals. He discusses how the company’s oligonucleotides platform is paving the way towards a brighter future, with the potential to target a wide range of severe and “undruggable” diseases, while overcoming drug delivery challenges and toxicity concerns. This interview was conducted on 4 October 2022. Updated December 2024. Interview with Dr. Monia, CEO of Ionis Pharmaceuticals Brett Monia (BM), Chief Executive Officer and Member of Board of Directors at Ionis Pharmaceuticals. Interviewed by Yalda Sedaghat (YS), Oligonucleotide Tx Project Leader, Evotec. Brett Monia serves as the Chief Executive Officer and a member of the Board of Directors at Ionis Pharmaceuticals, where he is also a founding scientist. His notable contributions include pioneering research in the medicinal chemistry and mechanisms of action of RNA-targeting modalities. Additionally, Dr. Monia holds a position on the Board of Directors for both Ionis Pharmaceuticals and Cognition Therapeutics. As a founding member of the Oligonucleotide Therapeutics Society (OTS), Dr. Monia has served as its President and on its Board of Directors. In 2025, he was honored with the year’s OTS Lifetime Achievement Award. In an interview with Yalda Sedaghat from Evotec, Dr. Monia recounted his journey in the field of oligonucleotide therapeutics and addressed several pertinent questions on oligonucleotide based drug discovery and development. About 6 The Journey to Becoming CEO YS: Could you please share some details about your background and educational experience? Specifically, what did you study and how did you end up at Ionis? BM: Sure, I got my PhD in pharmacology at the University of Pennsylvania. I always wanted to be in the drug industry, to discover and develop drugs, bring them to patients, do clinical trials, bring them to market, which is why I studied pharmacology. And as I was finishing up pharmacology, I then moved into a MD program at the University of Pennsylvania, and I was about to start my medical degree studies when I was approached by Stan Crooke, who was starting a new company in California that was going to develop a new platform of drug discovery called antisense. And I didn’t know too much about antisense at the time, but I read about it, and I realized that this will be very hard, but if it can work, it’ll really be a breakthrough for discovering new medicines for patients because it can deliver types of medicines that really no other platform would be able to do. So, I put my medical degree on hold, and I moved to California to help start ISIS Pharmaceuticals, which is now Ionis Pharmaceuticals. I started as a bench scientist and quickly took on more and more responsibility, expanded roles, I created a drug discovery group which I led for many years which is really responsible for the pipeline of drugs we have today and the marketed drugs we have today. And then my role expanded to include drug development, business development, and really all kinds of different areas at Ionis that were important to the company. It was about that time when the board approached me and asked me to consider moving into a CEO role. And that was because Stan Crooke was planning to retire and step off the board and step down. I wasn’t really that excited about it honestly. I didn’t see myself as a CEO, but maybe more of a head of R&D someday, but I thought about it. I talked to people; I got advice. The board was persistent. And I loved the company, and I felt like I could do things to really help the company be more and more successful. So, I moved into the CEO role in 2020, January 1st, and I’m now wrapping up my fifth year as CEO, and it’s been going very well for the company, and I’m really excited about where we are headed. Introducing ASO-Based Therapies YS: Ionis Pharmaceuticals is dedicated to the discovery and development of ASO (antisense oligonucleotide)-based therapies. Can you please tell us about Ionis’ approach and the thinking behind it? BM: So, the antisense platform is a revolutionary way, a new way, for discovering developing drugs for treating all kinds of diseases. Historically the pharmaceutical industry has focused on proteins. Small 7 molecule drugs that bind to proteins, or monoclonal antibodies that bind to proteins. And there’s been success there obviously, many successes. But the vast majority of diseases, chronic diseases in particular, still remain without any good treatment options today. Severe, lethal, fatal diseases that have no treatment options. And small molecules and antibodies haven’t delivered enough value. The other thing is that most of the drug targets that appear to be the right targets for these diseases where there is a high unmet need are not druggable with traditional approaches like small molecules or antibodies. That’s because the mechanism by which small molecules and antibodies work, they just can’t bind to or get to those proteins. Antisense, as I said, is a revolution because it doesn’t target protein. It targets RNA through Watson-Crick hybridization mechanisms and by targeting RNA, all targets are druggable theoretically. All you need to know is the sequence of the RNA and you could design your antisense drug and bind to that RNA and modulate the function of that RNA by either increasing the production of a protein that’s missing in a disease or decreasing the production of a toxic protein. And that’s how antisense works. And it works and is now a validated approach with many drugs on the market today, a rich pipeline of drugs that are approaching the market for severe diseases of all kinds. Overcoming Challenges in Oligonucleotide Delivery YS: Imagine for a second that delivery was not an issue anymore and we could get inside any cell type we wanted; do you think any gene can be modified by ASOs? BM: Based on the mechanism of action of antisense drugs, theoretically any gene product can be modulated, can be affected by an antisense drug … if you can get the drug there. If you can solve delivery. Delivery is the key really for all drug discovery platforms when you think about it. Antisense is no different. If you have the right chemistry, and you can deliver the drug to the cell type of interest, or if you can deliver the drug locally, like into the lung for lung diseases or the CNS for CNS diseases, you can theoretically modulate, block the production of a toxic gene product, regardless of the gene, any gene should be approachable as long as you can solve the delivery issue. YS: In the near future, do you think we will be lucky enough to find something like GalNAc for targeted drug delivery? “Ionis’s strong patientfocused culture is a key differentiator.” 8 BM: Something like GalNAc for other tissues you mean? YS: Yes, do you still think that we will be able to crack the code and achieve delivery to other tissues and is Ionis active in this area? BM: So, the LICA chemistry platform, really the start there was with targeting the liver, the hepatocytes. LICA stands for Ligand Conjugated Antisense, and it really has been a breakthrough for ASOs, antisense, as well as siRNA because it delivers the drug almost entirely to the hepatocyte, to the liver cells. And there is almost no exposure outside of the liver so that greatly increases the potency because you are not wasting drug for liver targets. It’s all getting to where it needs to go. And you’re not causing off-target side effects because you’re not exposing the drug to the rest of the body, you’re only going to the liver. That is GalNAc for the liver. We are developing other LICAs for other tissues today, and we’re making great progress. We have succeeded in a targeted delivery LICA approach for pancreatic beta cells where we can show very nice potency in animals by attaching a ligand to an antisense drug or an siRNA drug and it goes right to the beta cells in the pancreas and shows very nice target engagement. We’ve also done the same thing now for muscle, both skeletal muscle and cardiac muscle, cardiac myocytes where we have been able to identify ligands that target a particular receptor that is found in muscle cells, binds to those receptors, and the drug – siRNA or ASO – gets taken up into the cells where the drug exerts its effect very nicely. Very similarly to the liver in both of these cases. We are working on additional targeted delivery strategies, additional LICAs for other tissues and cell types, as well as similar approaches to overcome the blood brain barrier for CNS diseases. We are making good progress. Right now, the next two LICAs I see coming forward will be for muscle and to traverse the blood brain barrier for CNS diseases, supporting IV or SC dosing. A Spotlight on Ionis Pharmaceuticals YS: How do you see Ionis compared to other pharmaceutical companies? BM: Ionis is unique in several different ways. The two biggest ways: Number 1, the science, the platform, right? Obviously, RNA targeted therapeutics, ASOs and siRNAs, which we are doing both of at Ionis is a very new and revolutionary way to discover drugs that the pharmaceutical industry hasn’t been a part of at all in its history until recent. So purely from a drug discovery platform basis is where we differentiate from these other companies. The other thing that I think separates Ionis and makes it unique compared to other pharmaceutical companies is the culture. 9 The culture at Ionis is incredibly strong and it is a culture that is devoted to delivering transformational medicines for patients who are suffering from terrible diseases. And that is the primary driver. It’s not the economics, it’s not the financials around what the market could offer for a given drug. If we believe that we can help patients with a drug that we can discover, we will deliver it. We will do the best of our ability to deliver it. It’s also a great culture of transparency, dedication to innovation and science, and it’s an organization with a culture that is committed to empowering individuals to pursue their scientific innovative ideas without being harnessed, without being held back. And in a company with the fewest possible rules so that people don’t feel like they are cornered by too many rules, too many committees. We try to avoid bureaucracy at all costs. YS: Ionis has been very successful in CNS disorders with unprecedented therapies, to treat spinal muscular atrophy (SMA) for example. However, clinical trials for Huntington’s disease were paused. Can you please tell us about that? What could have been done to prevent it? Could Ionis have foreseen this prior to what we saw in the clinic? BM: So, the Huntington phase 3 trial last year was stopped. The Huntington trial tested our drug Tominersen which targets the huntingtin gene in patients with symptomatic Huntington’s disease whether they were early stage, middle stage, or late-stage disease. It was decided after all the patients actually enrolled in the Huntington trial, if I recall nearly 800 patients in that study, a safety oversight committee decided that patients were not going to benefit if the study completed, which would have taken another year, so they recommended to stop the study, and our partner Roche agreed. So, the study was stopped. What we showed in the study was very nice reductions of huntingtin protein in patients, but it wasn’t showing signs of benefit. Since that time, our partner Roche has investigated the data from that phase 3 study extensively, post hoc analysis and they believe they have found a patient population, a sub-population in that trial that was benefiting from Tominersen. That patient population are patients that have less disease burden. They are earlier on in their disease, and they are younger. It appeared that those patients were doing better across all endpoints that we looked at compared to placebo. Based on that analysis, Roche has started a new phase 2 study in that same exact population of Huntington’s patients to prove the hypothesis that if you can treat patients that are earlier on their disease with Tominersen, they can benefit. “Delivery is identified as a critical factor for RNA-targeted therapies” 1 0 That is another example of a good target, good drug, but a trial design that probably wasn’t perfect. YS: Why were the clinical trials designed that way? BM: Two reasons. Our preclinical data suggested that sure if you treat earlier, you can actually prevent the disease from actually occurring, before symptoms happen or you can even reverse them if you treat early enough. But also, animals that were further on in their disease progressed slower. So, they were getting some benefit even though they were still progressing, they were progressing slower than placebo. So, there was evidence that the different stages of the disease, patients with different stages of the disease would benefit. Just they would benefit differently. The second reason is that we wanted to help as many patients as we can. Patients that are early. Patients that are mid. Patients that are late. And all of those patients need treatment so those are really the two reasons we designed the trial that way. The Power of Oligonucleotide Chemistries YS: My next question is actually about the chemistry. The chemistry just keeps getting better every day and your generation 2.5 antisense drug has shown remarkable activity in vitro, in animal models and of course in the clinic and the market. Ionis has a few ASOs with cET, (constrained ethyl) modifications in clinical trials for metabolic disorders, but no marketed drug yet. Am I correct? Or are we getting close to that? BM: That’s correct. We have even better chemistries now entering development, such as our MsPA backbone chemistry which support very infrequent dosing; as infrequent as semi-annual or even annual dosing. YS: Other companies are developing copycat drugs using your old chemistry, 2′-O-methoxyethyl (MOE), mainly because the safety profile is known. What have you learned through all these generations of ASOs and siRNAs, and what workflows would you recommend for reaching the clinic with minimum to zero side effects? BM: Chemistries for antisense oligonucleotides and siRNAs continue to evolve and get better and better and better. New chemistries that are being developed now at Ionis are allowing us, for example, to remove phosphorothioate from the backbone. “By targeting RNA, all targets are druggable, theoretically. This is a breakthrough for discovering new medicines for patients.” 1 1 Phosphorothioate is a wonderful chemistry that supports RNase H and provides very effective distribution in the body. However, phosphorothioates also can produce pro-inflammatory effects at high concentrations in tissues. New backbone chemistries are now emerging at Ionis that allows us to remove the phosphorothioate from the backbone. And we have seen a really exciting profile with these new backbones. One backbone chemistry called MSPA as an example. When we incorporate MSPA into our oligonucleotides, and we really push the dose to produce pro-inflammatory effects, we are actually not seeing pro-inflammatory effects, so we are greatly reducing those off-target side effects. And what we are also getting from this backbone is a marked increase in stability, allowing us to dose much less frequently, potentially in the clinic. Every few months for example. Or twice a year for example. Because they are so stable. The other thing that is happening with new chemistries, and really this gets to another way to tackle off target effects and to avoid them is potency. The lower the dose, the better off you are, the less side affects you are going to get. Especially if you can get your drug to the cell type of interest. Newer chemistries, like gen 2.5 cET chemistry, the MSPA backbone, and of course that’s what LICAs do. LICAs for liver, for muscle, for pancreas, and other tissues. What they do is they allow us to go to much, much lower doses so that we can avoid off target effects and only deliver the drug to the cell type of interest and really that’s what it’s all about is to avoid chemistries that produce off-target effects and utilize chemistries that give you higher potency so that you can go to the lowest dose possible. That’s what cET brings. That’s what MSPA brings. That’s what LICA chemistry brings – greater potency, lower dose. YS: How about reducing the number of phosphorothioate from the backbone of ASOs and siRNAs? BM: We don’t remove all the phosphorothioate. We remove some of it. And that’s enough to avoid off target effects. By incorporating these MSPA backbone chemistries into them. So LNA chemistry like cET chemistry improves potency based on its ability to bind to RNA more tightly, higher affinity, right? But you raise a good point too is that we don’t have good ways to predict the best sequences still to develop an antisense drug or where to place some of these chemistries when we decorate the molecules, right? And a lot of this has to be done through screening. Hard, brutal screening. Screening lots of oligonucleotides with different decorations, different amounts of different chemistries in them in different sequences so you’ve got a lot there to screen. We have some idea. We have some rules. We have plenty of rules actually, but we still don’t have enough. And we still have to screen to find the right sequence and the right composition of chemistry that’s optimal to maximize potency and to avoid off-target effects. 1 2 Final Outlooks on Oligonucleotides YS: Ionis and Alnylam are key players in the field of RNA-targeted drug discovery and development. Both companies focus on creating innovative gene-based therapies. Of course, factors like delivery, specificity, tissue longevity, and safety are important. Do you see a clear winner here, ASO vs. siRNA? Example: the head-to-head ASO/ siRNA in the cardiovascular field for the same ATTR target, Eplontersen vs. Onpattro or Amvuttra. BM: I do not see the need to declare winners and losers. Both ASO and siRNA platforms have delivered a great deal of success and benefit for patients, and are poised to expand greatly on this in the future. Both mechanisms offer advantages and disadvantages. The key is to utilize the best mechanism, ASO or siRNA, to make the best drug for a particular target and disease. That is what we are doing at Ionis. YS: How about gene therapy, do you envision a future for that? BM: Traditional gene replacement therapy has had limited success to date, especially considering the many decades that this approach has been in development. However, I include gene editing as gene therapy, and maybe as the latest generation of gene therapy. Although there is much that needs to be done to validate this platform as a safe and effective approach in medicine, I am reasonably optimistic that this approach will have success. This is one reason why we are also investing in gene editing at Ionis. 1 3 When looking to develop innovative oligonucleotide therapies, partnering with a CRO/CDMO with specialist oligonucleotide capabilities can help you ensure success. Our integrated multidisciplinary team ensures that your oligonucleotide discovery and development program will progress efficiently from whatever entry point you require, up to and including regulatory submission. With more than 40 dedicated and experienced oligo experts, we will design and select state-of-the-art oligonucleotides, providing comprehensive preclinical data sets, reduced transition times and efficient communication. We can also help with disease area expertise and animal models to identify new targets or validate existing ones. Our computational chemists and bioinformaticians are involved in the early stages, with sequence selection and oligonucleotide synthesis. We then have a skilled set of biologists, pharmacologists, chemists, toxicologists, pathologists and regulatory experts to support screening and in vitro/in vivo proof of concept (PoC) right through to (and including) GLP safety studies and entrance into the clinic. We guide your programs through key deliverables, anticipated outcomes, project decision points and crucial stage handovers for oligonucleotides. Figure 1. Example of a timeline for a generic end-to-end oligo project. Partners can choose to join at any or all stages of the continuum, from target identification up to and including preclinical IND. End-to-End Oligonucleotide Platform Year 1 Year 2 Year 3 Year 4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Screen, H2L Rodent, early safety IND enabling tox + CMC In silico library design, synthesis and in vitro screening Safety assessment and de-risking activities go/ no-go decision point Preclinical tox and GMP manufacturing lead selection and resynthesis for vivo scale 3 4 2 5 1 In vivo target engagement PDC nomination IND filing 6 1 4 In silico Oligonucleotide Design In silico design focuses on identifying sequences with optimal drug-like profiles from a pool of potential candidates targeting a specific biomolecule. We have developed a proprietary pipeline to facilitate the selection of molecules exhibiting the most favorable combination of properties, enhancing the likelihood of identifying the best candidates during the screening process. Our pipeline is highly adaptable to the specific requirements of any project and includes the prediction of multiple features, such as: Off-target effects and conservation analysis across a wide range of species Toxicity assessments/presence of repeats Efficacy predictions and secondary structure analysis/thermodynamics parameters Our sequence design capabilities encompass siRNAs and ASOs with various mechanisms of action, including gene downregulation or upregulation, steric hindrance with RNA-binding proteins (RBPs), allelespecific downregulation, anti-MiR and exon skipping. Figure 2. A proprietary pipeline for generating and selecting the most promising oligonucleotide sequences with favorable drug-like properties. ASO selection no in-silico predicted off-target in humans no mismatch not conserved in macaque and not conserved in mouse not conserved in macaque no snps with MAF >0.1 no toxic motif all potential oligos of length 15 to 25 target transcript conserved in macaque not conserved in mouse conserved in mouse with ASO/siRNA design platform 1 Detailed target analysis 2 Generate ASOs/siRNAs of specified sizes 3 Align ASOs/siRNAs using BWA to: 4 Compute additional descriptors and proceed to the semi-automatic selection 1 5 Oligonucleotide Chemistry and Synthesis Following oligonucleotide design, We have a broad oligonucleotide synthesis platform that can fully support our partners’ projects. For the early phases of a discovery project, we have small-scale synthesis platforms based on the MerMade 48X Synthesizer. With a range of associated instrumentation, our platform can rapidly deliver single- and double-stranded oligonucleotide libraries for initial screening purposes, through to the synthesis of high purity lead compounds suitable for full in vitro and in vivo profiling. As a project matures, our mid-scale capabilities using the ÄKTA oligosynt™ platform can supply advanced leads and candidate oligonucleotides on scales from 100s of milFigure 3. Evotec scientist performing small-scale synthesis on the MerMade 48X Synthesizer. ligrams up to 10s of grams to support efficacy, tolerability and toxicology studies in rodents and higher species. Our analytical chemistry teams are equipped with the latest high-resolution LC-MS systems to ensure the highest quality of delivered compounds. A range of complementary characterization techniques are available, in addition to the generation of analytical packages to support advanced studies including packages for entry into preclinical development and regulatory support. Our synthesis experts are able to produce a wide range of oligonucleotide classes, from single-stranded antisense gapmers and mix- 1 6 Figure 4. Evotec scientist desalting the oligo. Figure 5. An overview of Evotec’s oligonucleotide synthesis capabilities, including milligram and gram scale production and the upcoming GMP scale manufacture. mers through to double-stranded siRNAs and miRNAs, incorporating a variety of industry standard and project specific chemical modifications to the backbones, sugars and nucleobases. We work with commercial building blocks from multiple suppliers and can also synthesize custom reagents for novel oligonucleotide modifications according to project and client needs. A wide range of conjugation chemistries are also available to improve cellular uptake or enable tissue and organ targeting. In addition to well-known and validated strategies for improving targeting and uptake such as GalNAc, lipid and cholesterol conjugates, etc. with dedicated and well-equipped chemistry teams, we have expertise and a strong track record in linker chemistry and peptide synthesis. We also offer a wide range of capabilities for conjugation, antibody generation and small molecule synthesis. Other chemical classes can also be harnessed to expand the chemical scope of oligonucleotide therapeutics and accommodate a project’s individual needs. Current Platform Capabilities in Oligonucleotide Synthesis Milligram scale Gram scale GMP Manufacture (0.5 kg) Future platform expansion Wide range of chemistry capabilities – Phosphate and phosphorothioate chemistry – Fully modified sugar chemistry (2’-F, 2’-OMe, 2’-MOE, LNA, others.) – Nucleobase modifications (5-MeC 5-MeU etc.) – Wide range of building blocks available from multiple suppliers – Conjugation chemistry (GalNAc, lipids, dyes, ASO-peptides conjugates) – In-house building block/ linker/ligand synthesis available Small scale equipment and capacities: 2 complete Oligo discovery platforms in Evotec Medium scale equipment and capacities: Gram scale to support preclinical studies AKTA Flux 6 (Tangential Flow filtration) SP VirTis AdVantage Pro Freeze Dryer – AKTA Oligosynt – Latest automated system for research to process development scales (50 µmol to 12 mmol) Post synthetic processing Post synthetic processing (in addition to small scale) Synthesis Synthesis MerMade 48X (Automated parallel synthesis from 200 nmol to 5 µmol) Agilent 1260/1290 Infinity II (HPLC Purification) AKTA Pure (Desalting) Purification Thermomixer for strand annealing Nanodrop Concentration determination Genevac evaporation/ freeze drying Endosafe Endotoxin titration AccurateMass LC/MS 1 7 Conjugation of therapeutic oligonucleotides to ligands and biomolecules can improve tissue/organ targeting and uptake A range of molecule classes are being exploited or investigated in the industry including: – Sugars/carbohydrates – e.g. GalNAc for liver targeting – Lipids – e.g. C16 or cholesterol for liver, kidney, adipose, CNS – Peptides, e.g. GLP-1 analogues for pancreas – Antibodies – e.g. TFR1 mAB for skeletomuscular tissue Wide range of conjugation chemistries used in Evotec/Partner projects – Lipids, carbohydrates, dyes, peptides, biotin etc. – 5’ and 3’ modifications for all ASOs – Passenger strand modifications for siRNA – Cleavable and non cleavable linkers – Organic chemistry capabilities for synthesis of novel or non-commercial conjugate and linker building blocks GalNAc ASO/siRNA conjugates Dye labelled ASOs Peptide – ASO conjugates siRNA – antibody conjugates Lipid – Oligo conjugates Figure 6. Improving targeted ASO delivery by using ligands and biomolecules. 1 8 Efficacy (Target Engagement in vitro) ASO Screening and Profiling Oligonucleotides can modulate mRNA expression or splicing, leading to altered protein expression and potentially reversed disease phenotypes. We design customized screening cascades to monitor each step of this process, ensuring the selection of the most potent and safest oligonucleotides. We have developed a miniaturized (384- well), multiplexed, one-step RT-qPCR using proprietary reagents to measure target expression modulation (down- or up-regulation) or splicing modification of over 1,000 oligonucleotides in under a month. Hits are confirmed through concentration-response testing to identify the most potent ASOs. Hit validation is performed by assessing downstream effects, such as protein level modulation and/or functional assays. Various technologies, including high-content imaging, ELISA, MSD, TR-FRET and Simple Western, are employed for protein level analysis. The functional effects of oligonucleotides are evaluated using customized cellular assays in both healthy and diseased contexts, depending on the target function. We offer a wide array of cell models, including immortalized cell lines, primary cells, iPSC-derived cells and patient-derived cells for genetically based diseases. Our iPSC platform, combined with gene editing (e.g., CRISPR technology), ensures that oligonucleotides are tested on disease-relevant cell systems. Figure 7. Typical workflow of in vitro discovery activities for oligonucleotide therapeutics projects. High-throughput oligonucleotide screening (RNA readout), followed by protein analysis and/or phenotypic assays using diseaserelevant cell models to identify the most potent ASOs. Screening and cell treatment – ~1000 oligos throughput Protein analysis Phenotypic and disease-relevant assays Expression of target (and relevant isoforms) Optimization of oligonucleotides delivery according to modality and conjugation strategy Automated handling, storage and compound plates preparation Echo dispenser RNA analysis qPCR read-out – 1-Step qPCR protocol – Multiplexed qPCR High-throughput Genedata Screener analysis – Western Blot and Simple Western (Jess) – High Content Screening (HCS) Operetta CLS – Meso Scale Discovery (MSD) Simple Western Jess High content imaging Operetta CLS Metabolic studies Seahorse FX Functional studies FLIPR Penta Other assays – High content imaging – Electrophysiology – Pathway analysis – Biochemical assays Cell treatment up to 384w format 1 9 Figure 8. Examples of cells employed for in vitro profiling of oligonucleotides, ranging from iPSC-derived cell models (either 2D or 3D) to patient-derived cells for splicing correcting oligonucleotides. 2 0 In vitro Safety A crucial aspect of antisense therapeutics discovery is early safety monitoring in the preclinical phase to predict potential toxicities. We conduct medium-throughput toxicity prediction assays during hit validation to mitigate risks as oligonucleotides progress through the screening cascade. Established assays for predicting hepatotoxicity, immunogenicity, and neurotoxicity utilize well-characterized reference ASOs from preclinical models or clinical settings. Hepatotoxicity is monitored via caspase pathway activation, while immunogenicity is assessed through TLR9 activation in a human lymphoma cell line. For selected leads, cytokine release in freshly prepared PBMCs from multiple healthy donors is tested to confirm the lack of immunogenicity in a clinically relevant setting. Prediction of Hepatotoxicity (Caspase 3/7) To assess the potential hepatotoxicity of the selected antisense oligonucleotides, we employ a Caspase 3/7 assay as described by Dieckmann et al. (2018)1 and Shen W et al. (2019)2. Human (HeLa) and murine (AML12) cells are used for prediction of potential hepatoxicity in human and mouse, respectively. The use of human and murine models helps two-fold: prediction of hepatoxicity in human and de-risking of following in vivo studies in rodents. The results show caspase 3/7 activation and indicated that the toxic control ASO LN43 induced caspase activation, whereas neither the non-toxic control ASO LN32 nor the test ASOs triggered such activation. References 1 Dieckmann A, Hagedorn PH, Burki Y, et al. A Sensitive In Vitro Approach to Assess the HybridizationDependent Toxic Potential of High Affinity Gapmer Oligonucleotides. Mol Ther Nucleic Acids. 2018;10:45-54. doi:10.1016/j.omtn.2017.11.004 2 Shen W., De Hoyos C. Migarawa M. et al. Chemical modification of PS-ASO therapeutics reduces cellular proteinbinding and improves the therapeutic index. Nature Biotechnology 2019 37:6. 10.1038/s41587-019-0106-2 Figure 9. Caspase-Glo® 3/7 Assay showing the induced caspase activation with the toxic control, ASO LN43 in human and murine cell lines. Cells are transfected 5 concentrations ranging from 100 nM to 1 nM Molarity for 24 hours Luminescence fold increase vs. UT Human Cells -10 16 14 12 10 8 6 4 2 0 -10 -9-9 -8-8 --77 --66 0 2 4 6 8 10 12 14 16 HeLa cells Molarity Luminescence fold increase vs. UT -10 -9 -8 -7 -6 0 2 4 6 8 10 12 14 16 AML12 cells Molarity Luminescence fold increase vs. UT LN43 LN32 UT Murine Cells Molarity Luminescence fold increase vs. UT -10 16 14 12 10 8 6 4 2 0 -10 -9 -8 -7 -6 -9 -8 -7 -6 0 2 4 6 8 10 12 14 16 HeLa cells Molarity Luminescence fold increase vs. UT -10 -9 -8 -7 -6 0 2 4 6 8 10 12 14 16 AML12 cells Molarity Luminescence fold increase vs. UT LN43 LN32 UT LN43 LN32 UT -10 -9 -8 -7 -6 0 2 4 6 8 10 12 14 16 HeLa cells Molarity Luminescence fold increase vs. UT -10 -9 -8 -7 -6 0 2 4 6 8 10 12 14 16 AML12 cells Molarity Luminescence fold increase vs. UT LN43 LN32 UT 2 1 Prediction of Immunotoxicity (Bjab and hPBMCs) The in vitro immunogenicity assays on the Bjab cell line and human peripheral blood mononuclear cells (hPBMCs) are essential for the evaluation of potential inflammatory risk associated with ASO treatment. We developed these predictive assays after in vivo studies on rodents and non-human primates were unable to predict the immunogenicity of some ASOs1. Instead of animal models, these assays use Bjab cells (Burkitt lymphoma cell line) or fresh hPBMCs (coming from our blood bank service that guarantees fresh blood from healthy volunteers). The Bjab cell line has been described to respond to ASOs that showed immunogenicity in the clinic with a CCL22 mRNA increase (not elicited by non-immunogenic ASOs), through TRL9-dependent signalling2. The model has been demonstrated to robustly predict potential ASO immunogenicity. The Bjab assay is validated and performed in a high throughput format to allow assessment of a larger number number of ASOs for an immunogenicity prediction at hit validation stage (Figure 10). Only freshly isolated hPBMCs seem suitable compared to cryopreserved cells1 and are used to assess the potential risk of proinflammatory cytokines modulated by ASOs in a complex and translational model, where different networks involved in the recognition of both DNA and RNA oligonucleotides, including the Toll-like receptors (TLRs), are present. After treatment of Figure 10. Fold change CCL22 mRNA induction in Bjab cells of positive and negative ASO clonical controls. Data are expressed as fold change relative to untreated cells ± standard deviation.2 hPBMCs with ASOs, cytokine concentrations are measured in supernatants by the Intelliflex xMAP™ instrument with multiplexed approach (Figure 11). Viability is analyzed in pellets. During our assay development process, several cytokines and molecular tools were tested to ensure a robust readout that is benchmarked to relevant positive and negative clinical controls. The difference between clinically relevant positive and negative control ASOs demonstrates that this assay is sufficiently sensitive to distinguish between immunogenic and non-immunogenic ASOs (Figure 11) despite donor-to-donor variability and subtle pro-inflammatory effects that might otherwise be overlooked during preclinical and could translate into significant toxicity in clinical trials.2 1×10-8 100 75 50 25 0 1×10-7 1×10-6 1×10-5 [ASO] Fold change CCI22 (rel to UT) 1×10 -8 1×10 -7 1×10 -6 1×10 -5 0 25 50 75 100 [ASO] Fold change CCl22 (rel to UT) UT ASO positive clinASO negative clUT ASO positive clinical control ASO negative clinical control 2 2 Cell viability, donor 2 Cytokine, donor 1 Cytokine, donor 2 Figure 11. Top graphs: Cell viability assessed in cell pellets after treatment with dilution curves of reference ASOs in two donors. Data are expressed as relative luminescence unit (RLU) ± SEM. Middle and bottom graphs: Cytokine release in supernatants from the same ASOs tested with the Luminex assay. The graphs on the right are an enlargement of those on the left, given that positive technical control shows higher cytokine release compared with other clinical ASOs. Data are expressed as pg/mL of cytokine ± SEM. References 1 Burel SA, Machemer T, Baker BF, et al. Early-Stage Identification and Avoidance of Antisense Oligonucleotides Causing Species-Specific Inflammatory Responses in Human Volunteer Peripheral Blood Mononuclear Cells. Nucleic Acid Ther. 2022;32(6):457-472. doi:10.1089/nat.2022.0033 2 Pollak, A., Cauntay, P, Machemer T., et al. Inflammatory Non-CpG Antisense Oligonucleotides Are Signaling Through TLR9 in Human Burkitt Lymphoma B Bjab Cells. Nucleic Acid Ther. 2022, 10.1089/nat.2022.0034 3 Vollmer J, Weeratna R, Payette P, et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol. 2004;34(1):251-262. doi:10.1002/eji.200324032 Neg ctrl Technical ctrl ASO positive clinical ctrl ASO negative clinical ctrl Neg ctrl Technical ctrl ASO positive clinical ctrl ASO negative clinical ctrl Neg ctrl Technical ctrl ASO positive clinical ctrl ASO negative clinical ctrl Cytokine, donor 1 Cytokine, donor 2 Luminescence [RLU] Cytokine conc [pg/mL] Cytokine conc [pg/mL] Cytokine conc [pg/mL] Cytokine conc [pg/mL] Log ASO conc [M] Log ASO conc [M] Log ASO conc [M] Log ASO conc [M] Log ASO conc [M] Cell viability, donor 1 Luminescence [RLU] Log ASO conc [M] 2 3 Prediction of Acute Neurotoxicity for CNS Applications – Multielectrode Array (MEA) The development of ASOs for central nervous system administration often encounters high attrition rates, due to acute neurotoxicity. This neurotoxicity is influenced by both the nucleotide sequence and chemical modifications, and it remains largely unpredictable during the design phase. We have established an in vitro screening assay utilizing the Multielectrode Array (MEA) to characterize these neurotoxic oligonucleotides. MEA systems facilitate the study of electrically active cells, such as neural networks. Our observations indicate a strong correlation between specific assay parameters and neurotoxicity, enabling us to effectively exclude neurotoxic ASOs from further in vivo development. Figure 12. Various intracellular and extracellular approaches to record neuronal electrical activity with a multielectrode array (MEA) system.1 Patch clamp Neuron Substrate Microelectrode Array (extracellular field potential) Extracellular (Action potentials) Intracellular (transmembrane potentials) Figure 13. Multielectrode array (MEA) approach2 (A) Schematic of chamber set-up. (B) Representative image of cultured neurons (14 DIV) grown on MEA. (C) Single electrode recordings showing examples of different firing patterns. Scale bars 30 μV. A B C 2 4 References 1 Mateus JC. Optimization of a multielectrode array (MEA)-based approach to study the impact of Aβ on the SH-SY5Y cell line. 2015. https://api.semanticscholar.org/CorpusID:139373792 2 Roberts TP, Kern FB, Fernando C, et al. Encoding Temporal Regularities and Information Copying in Hippocampal Circuits. Sci Rep. 2019;9(1). doi:10.1038/s41598-019-55395-1 3 Mack CM, Lin BJ, Turner JD, Johnstone AFM, Burgoon LD, Shafer TJ. Burst and principal components analyses of MEA data for 16 chemicals describe at least three effects classes. Neurotoxicology. 2014;40. doi:10.1016/j. neuro.2013.11.008 4 https://axionbiosystems.com/maestro-and-omni The impact of these ASOs on the functional properties of the neuronal network is then assessed at various time points. Electrophysiological parameters that show strong correlation with in vivo neurotoxicity are analyzed to identify potential neurotoxic ASOs. This assay facilitates the early identification of neurotoxic ASOs, thereby reducing attrition rates during in vivo tolerability studies. Figure 14. Microelectrode Array (MEA) neurotoxicity assay. A) MEA well-containing electrode grids on which cells are cultured. B) Example recorded neuronal activity showing threshold for detecting action potentials 3, 4. C) Test ASOs are added on DIV13 and their effects are recorded after 1h, 2h, 4h and 24h post-treatment. D) Effect of ASOs on selected electrophysiological parameters. A D Burst Spike Detection Threshold B C Control Test ASO Not Toxic Parameter 1 Undefined Parameter 1 Toxic Parameter 1 Parameter 5 Parameter 2 Parameter 4 Parameter 3 Parameter 5 Parameter 2 Parameter 4 Parameter 3 Parameter 5 Parameter 2 Parameter 4 Parameter 3 +ASO Recording neuronal activity after 1, 2, 3, 4, 24 hr Mouse cortical primary neurons + Astrocytes DIV 13 2 5 Off-Target Analysis Figure 15. Sophisticated transcriptomics platforms to derisk off-target effects. Mitochondrial stress Complement activation Pathway analysis Standardized semiautomatic process for sample avoids bias Standardized tissue sampling protocol Mus musculus/Rattus norvegicus LN2 RNAlater From tissue sample to ready-to-sequence library within one week PanHunter sophisticated analysis for in vitro or in vivo transcriptomics analysis Compatible with various tissue storage solutions Barcoded sample tracking in 96-well format RNA extraction RNA QC Automated reformatting ScreenSeq Qualitative Quantitative Barcoded vials and semi-automated workflow for sample tracking Target KD Target KD per cell type 2 6 PK and PK/PD Characterization of Oligonucleotides Pharmacokinetics (PK) and pharmacodynamics (PD) characterization are essential to predict how drugs including oligonucleotides will behave in the body, helping to ensure a smooth transition from preclinical to clinical development. PK/PD strategies include in vitro ADME studies and translational in vivo animal models. We provide comprehensive support for the in vitro ADME and in vivo characterization of oligonucleotides, through a variety of in vitro assays and in vivo models. This work is underpinned by extensive bioanalytical expertise across multiple modalities. The data generated are utilized by our expert modelers to evaluate the pharmacokinetic and pharmacodynamic relationships of oligonucleotides, facilitating accurate human predictions from the discovery phase through to development. The ability to have accurate pharmacokinetic and pharmacodynamic data are an essential component of a solid PK/PD translational model. Figure 16. PK and PK/PD strategies, including in vitro and in vivo studies, underpinned by extensive bioanalysis and PK and modeling. 1 for ASO; 2If on path/if requested. Solid track record for successful application of PK and PK/PD strategies in different therapeutic areas Numerous scientists with accumulated experience to implement in vivo PK and PK/PD translational studies from target validation to development phase In vitro Characterization A range of in vitro assays: – Plasma Protein binding1 – Plasma/tissues stability – MetID – Early in vitro tox. (cytotoxicity, mitochondrial toxicity, etc.) – CYP induction2; CYP and transporters inhibition2 In vivo PK/biodistribution – Rodents (including transgenic mice) and NHPs – Availability of a range of existing animal models/ administration routes in different therapeutic areas – Development of novel animal models PK, PK/PD modeling PK assessment and PK/PD modeling capabilities to allow translation from preclinical species to humans, from discovery to development phase Bioanalysis Unique bioanalytical platform, including LC/MS-MS, QuantiGene, MSD, Splint Ligase and Imaging (IVIS), to support in vivo PK and PK/PD studies LC/MS-MS QuantiGene Splint Ligase Imaging 2 7 in vivo Studies Following in vitro screening, we continue with an in vivo program to progress to clinic. We have full in vivo capabilities to support oligo projects with determining the good therapeutic window. For this aim, the in vivo safety and in vivo proof of concept including target engagement or efficacy on disease models, will be evaluated. The in vivo screening strategy for ASOs will be adapted to the major risks of each project. The therapeutic area is a main driver of the chronology of the various studies. As an example, for CNS projects, the acute tolerability following Intra cerebro ventricular or intrathecal administration will be the first risk to evaluate whereas for systemic administration the target engagement in the organ of interest will be the primary read-out. The choice of the screening cascade which will be proposed is led by the complexity of the studies. As an AAALAC accredited company and in the spirit of the 3Rs (replacement, reduction and refinement), we will propose to maximize the amount of information from each in vivo study. To be rapidly evaluated for the correlation between the concentration and safety/efficacy ratio, we will specifically design protocols for safety assessment, target engagement and exposure. The translational PK/PD evaluation remains present during the whole process of the in vivo evaluation. For safety assessment, we have developed a complete panel of read-outs to mitigate risk: General or specific clinical observation as neurological behavioral assay Histology and associated pathology Clinical chemistry Specific biomarkers including evaluation of hepatotoxicity and nephrotoxicity The discovery safety proposal will guide the project through to GLP IND enabling toxicology studies. For Proof of concept, you will benefit from the therapeutic area expertise of our scientists. This includes the use of sophisticated human-relevant disease models, such as humanized mice. If sequence homology allows, target engagement in relevant target tissues is conducted. This is fully supported by experts in all standard routes of administration and is complemented by a suite of bioanalytical techniques applicable to oligonucleotide discovery, including LC-MS/MS, RNAscope, MSD, hELISA, IHC, H&E, QPCR, etc. in vivo studies can be conducted in all relevant rodent species and NHPs Tailored oligonucleotide-specific in vivo studies for optimal PK/PD and efficacy readouts Humanized mouse models (in-house or purpose-bred) for PK/PD studies Multiple animal disease models and expert biology support from dedicated therapeutic areas including Metabolic Disease, Oncology, Infectious and Immune, CNS, Pain and Neurology 2 8 Intravenous (IV) Intracerebroventicular (ICV), Local bolus injection Gradual administration via mini pump Intrathecal (IT) Intraparenchymal (IP), Subcutaneous (SC) Rodent tolerability studies are included with all routes of administration, including prolonged formulations, inhalation, intra-vitreal, icv, iv, subQ, perOs, intratracheal and intrathecal In-depth analysis, including LC-MS/MS (PK), qPCR and MSD (PD), multipleomics analysis (off-target analysis, pathway analysis, and early toxicity analysis) and pathophysiological examination (early tox analysis) These industry-leading capabilities ensure partners transition from preclinical to clinical development with high confidence in their compounds. Figure 17. Example routes of administration with rodent tolerability studies. Bioanalytical Capabilities Bioanalysis is critical during both preclinical and clinical development to characterize candidate oligonucleotides in biological samples. We have developed strong bioanalytical expertise to support multiple modalities including the chemical complexities of oligonucleotides and oligonucleotide conjugates (Figure 18). Depending on the oligonucleotide type or its development stage, an array of bioanalytical techniques can be employed, including LC-MS/MS, SplintR Ligase qPCR, QuantiGene™ and SMxPRO/MSD (Figure 19). In addition to the above-mentioned quantitative methods, we have the possibility to use semiquantitative and qualitative methods that allow us to visualize the distribution of an oligonucleotide down to the cellular level, like high content quantitative image analysis (multiplex of immunohistochemistry and in situ hybridization). The techniques for each oligonucleotides need to be chosen based on the type of matrix analyzed, development stage, sequence complexity, required sensitivity and possibility to perform a bioanalytical assay in a regulated environment, following ICH and the Regulatory Agencies’ (EMA, FDA etc.) recommended best practices. 2 9 Figure 19. An array of bioanalytical techniques supporting oligonucleotide development. Figure 18. Complex structures and chemistry: combination of aromatic bases, polar sugars and polyanionic phosphates SplintR qPCR – QuantStudio 7pro™ Real Time applicable for hybridization-based gene expression assay – Higher sensitivity with an increase in the length/size of the Oligonucleotides – Semi-automated and easy sample preparation – High sensitivity Hybridization ELISA – Applicable for Oligonucleotides above 15 mer using hybridization – Applicable to plasma/serum, more challenging in tissues due to matrix effect – Sensitivity and Dynamic range higher with ECL detection; reduced matrix effect Whole body imaging Fluorescent based imaging (IVIS) – Non-destructive, use of fewer animals – Temporal description of biodistribution of compounds in the same animal – Can be used to assess lipid nanoparticles biodistribution Whole Body Section Fluorescence Imaging (WBSFI) – Analogous to whole body autoradiography (QWBA) but using fluorescently labelled test compound. – High-resolution images – Evaluation of several organs/tissues in the same sample – Semi-quantitative – Only total fluorescence (drug itself and its metabolites or its degradation products) can be measured – May be possible on larger animals than mice (rat and NHP) – Preliminary biodistribution evaluation LC-MS/MS UPLC coupled with triple quadrupole/qToFor Xevo TQ-S mass spectrometry – Oligonucleotides up to 40-50 mer QuantiGene – Hybridization-based gene expression assay that utilizes branched DNA for signal amplification. – Quantification of Oligonucleotides. MSD – Plate based immunoassay on the MSD – Oligonucleotides using hybridization of labelled oligos utilized as capture and detections probes. Linker & Conjugate GalNAc conjugates (platform-based approach) – Alnylam Pharmaceuticals with Enhanced Stabilization Chemistry (ESC+) – Dicerna’s GALX platform – Arrowhead’s TRiM platform Lipophilic conjugates – Olix (cp-asiRNA: 3’-cholesterol for permeation) – Phio (sd-rx-RNA: 3’-cholesterol for self-delivery) – Arrowhead’s DPC platform (Dynamic PolyConjugate) with cholesterol Peptide-conjugates formulations – Siranomics: polypeptide nano particules (PNP) targets liver and tumors Phosphorothioate (PSP) Fomivirsen Ionis/Novartis First Generation ASO PS +LNA Miravirsen Santaris PMO Eteplirsen Sarepta Golodirsen Sarepta Viltolarsen NS Pharma Third Generation ASO 2’O-methoxylethyl (2‘-MOE) Inotersen Akcea Volanesorsen PS + 2‘-MOE Nusinersen Ionis/Biogen Mipomersen Ionis/Genzyme Milasen BCH Second Generation ASO 3 0 Quantitative Methods LC-MS/MS Developing methods for analyzing new oligonucleotides is a complex process that often requires an array of technologies to Figure 20. An LC/MS assay workflow using a multi technique approach for sample preparation Modality MW Technique Samples Bioanalytical capabilities ASOs, siRNAs or Oligoconjugates 6–15 KDa Multi technique approach – Protein Precipitation Cold Ethanol, Methanol, Acetonitrile mixtures and ion pairing agents – LLE (Phenol/chloroform), – SPE: WAX or HLB micro elution plates – Multiple combination of ppt, LLE and SPE – Plasma and CSF (rat, mouse, NHP) – Tissue homogenates [Lung, Kidney, Liver, Bone marrow, fat tissues (WAT, BAT), ovaries, spleen, brain, spinal cord, heart, muscles] – Different buffers system – Hepatocyte assay – HUPLC IP-RP (HFIP + Alkyl amines) – Triple Quadruple MRM – qToF high resolution mass spectrometry – Internal standard – oligos analogue Method development Use of generic workflow Optimization of sample extraction and chromatography Fast turnaround (up to 10 Oligos) Bioanalysis Precision and accuracy (Typical LLOQ 5 ng/mL) Support to in vitro and in vivo studies with easy method transfer Area ratio Analyte concentration (ng/mL) 10,000 15,000 20,000 25,000 5,000 0 18 16 14 12 10 8 6 4 2 0 test various sample extraction and analysis approaches. 3 1 One of the primary challenges in liquid chromatography-mass spectrometry (LCMS/MS) analysis of oligonucleotides is the presence of negatively multicharged species. This reduces assay sensitivity compared to assays involving singly charged small molecules. Additionally, the negatively multicharged backbone of oligonucleotides tends to adhere to disposable materials and instrument components, leading to adsorption/desorption processes that result in compound loss, carryover and assay non-linearity. The high polarity of oligonucleotides also contributes to their poor retention in reversed-phase chromatography, necessitating the use of ion-pairing agents to achieve adequate chromatographic retention. This introduces further complexity, as ion-pairing reagents can bind to instrument components, potentially affecting subsequent analyses performed on the same instrument. Therefore, it is advisable to dedicate specific instruments to these assays. To better manage these challenges, we employ bio-inert ultra-performance liquid chromatography (UPLC) systems coupled with high-resolution mass spectrometry. Combining sample preparation with high-resolution instrumentation allows to achieve quantification limits in the low ng/ mL range. SplintR Ligase qPCR Quantification of oligonucleotide therapeutics in preclinical and clinical settings with high precision and sensitivity is essential to better elucidate their function, to facilitate clinical translation and to support clinical studies. Currently, several hybridization-based methods have been developed to increase sensitivity and throughput of the current gold-standard LCMS method. Among these, the SplintR ligase qPCR method has been shown to possess high sensitivity, throughput and to be cost effective.1 The method involves hybridization of probes and target oligonucleotide, subsequent enzymatic ligation by SplintR enzyme and quantification by qPCR using the absolute quantification method (Figure 22). Figure 21. Evotec LC-MS/MS approach to streamline bioanalysis based on project stage. Up to 10 ASOs in many tissues Single matrix for calibration & matrix effect – potential bias assessed for all other tissues One method that fits all for fast results Up to 3 ASOs in selected matrices Each ASO quantified against a proper curve (1 ASO - 1 Matrix approach) Batch number reflects number of ASO x number matrix Screening method Ad-hoc method 3 2 In-house design of DNA-based probes allows for a fast and cost-effective process. Probe set composition of 2 probes increases specificity in qPCR readout. Hybridization and enzymatic ligation of annealed oligo-probes in biological matrixes occurs in one-mix reaction using PCR thermocycler. The ligation product is the template of qPCR readout for ASO quantification References 1 Shin M. et al., NAT, 2022: Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction). using the absolute quantification standard curve method (Figure 22). SplintR ligase qPCR has shown high sensitivity and throughput with a broad linear range (ng/ml to fg/ml) in difference tissues and biofluids as well as low sample volume required. Figure 22. SplintR ligase qPCR workflow: probes-oligo hybridization and enzymatic ligation via SplintR ligase followed by qPCR of ligation product for oligo quantification via absolute quantification standard curve method. Modality MW Technique Samples Bioanalytical capabilities Oligonucleotide Therapeutics: ASO 4–8 KDa Hybridization-based method – Probes design for target ASO: In-house design and selection – Single enzymatic ligation step: probes ligation with SplintR ligase enzyme – qPCR quantification step: absolute quantification of ASO by standard curve method – Plasma and CSF (mouse, cynomolgus monkeys) – Tissue homogenates (Kidney, Liver) – 96w and 384w format workflow – Automated liquid handling with acoustic Echo – PCR thermal cycler – QuantStudio 7 Pro Real-Time PCR Systems Method development In-house probe design and selection Linear range definition Matrix effect evaluation Bioanalysis Quality controls (QCs) identification and validation Support to in vivo studies High sensitivity and throughput Low cost Ct values Molarity (log scale) 10-14 10-7 10-8 10-9 10-10 10-11 10-12 10-13 35 30 25 20 15 10 5 0 3 3 QuantiGene™ The QuantiGene™ Singleplex Assay Kit technology relies on a hybridization-based gene expression assay that uses branched DNA for signal amplification. It allows direct quantitation of RNA, DNA and microRNA using probe sets and can be adapted for siRNA or ASOs. It can be used for quantification in serum, plasma, tissues, or cell lysates. This technology is highly sensitive and may be useful to quantify very low concentrations in study samples, but it is not specific (parent and truncated metabolites). As the technology relies on the use of probe sets, it is more applicable for oligonucleotides with sizes larger than 16 nucleotides (nt). Indeed, it might be complicated to quantify ASOs shorter than 16 nt, as there can be potential difficulties that prevent optimal binding. Figure 23. Quantigene method development for oligonucleotide quantification. Modality MW Technique Samples Bioanalytical capabilities ASOs > 16-20 mer (optimal binding for shorter ASOs is challenging due to reliance on two probes) – Hybridization-based gene expression assay that utilizes branched DNA for signal amplification. – Quantification of specific RNA molecules based on specific QuantiGene™ probes. – Readout: luminescence – Plasma and CSF (rat, mouse, NHP). – Tissue homogenates [liver, kidney, brain, heart, adipose tissues]. – Different buffers systems depending on the matrix to analyse. QuantiGene™ Method development Probes design Can be performed on specific oligos sequences Bioanalysis Precision & accuracy (LLOQ down to pg/mL) Supports in vivo studies where high sensitivity is required Small sample size (5 to 160 µL depending on minimum sample dilution & 6 to 25 mg) Log10 (signal) Log10 concentration Example of calibration curves 0.0 0.5 1.0 1.5 2.0 2.5 7.5 7.0 6.5 6.0 5.5 5.0 4.5 https://www.thermofisher.com/de/de/home/life-science/ gene-expression-analysis-genotyping/quantigene-rna-assays.html Label Probe Amplifier Pre-Amplifier Target Probe - A mix of oligos binding to specific RNA target - Each pair of oligos secures one bDNA “tree” RNA 3 4 ASO Immunodetection Techniques SMxPRO/MSD In developing novel oligonucleotide-based therapeutics, the reliable detection and quantification of ASOs in experimental and clinical specimens are essential for understanding their mechanisms of action and transitioning from preclinical to clinical research. This necessitates the use of specific assays to detect ASOs in complex biological matrices. One such robust and sensitive method is the hybridization-based immunoassay, which enables the quantification of individual ASOs. This method involves designing specific oligonucleotides complementary to the ASO sequence (Figure 24). A biotinylated capture oligonucleotide and a DIG-conjugated detection oligonucleotide simultaneously anneal to the target ASO in the biological matrix under denaturing conditions using a PCR cycler. Incorporating locked nucleic acid (LNA) nucleotide modifications enhances the hybridization affinity of the analyte, thereby improving sensitivity, specificity and robustness. Quantification of ASOs is then achieved through immuno-based detection of the formed hybrid (Figure 25). Hallmarks of ASO immuno-based detection assay: High sensitivity Wide dynamic range (4–5 logs) Little matrix effects Figure 24. Design of oligo probes for ASO immunodetection assay on MSD or SMC platform. Figure 25. Principle of ASO immunodetection assay on MSD or SMC platform. Hybridization reaction Immunoassay-based detection Simultaneous hybridization of probes to ASO target under denaturing conditions (on PCR cycler) Biotinylated capture probe Digoxygenin detection probe ASO-probes hybrid SMCxPRO For CSF/Plasma - Ultra-high sensitivity - Single molecule counting - Reliable and suitable for clinically relevant matrices MSD S600 For tissue homogenates - High throughput and sensitivity - ELISA-like, well customizable - Very robust ASO Hybrid binding Hybrid capture on bead Detect Elute Quantify (laser detection) Immunodetection 3 5 The hybrid is typically quantified using traditional ELISA or the more robust Meso Scale Discovery (MSD) platform1. We have also developed a protocol for the SMC (single molecule counting) platform, which offers significantly higher sensitivity. For MSD assay applications, the hybrids bind to a streptavidin-coated plate during the first incubation step, followed by detection using an ST-labeled anti-DIG antibody and readout on an MSD reader. In contrast, our advanced SMC assay captures the hybrid on coated magnetic beads and detects it with fluorescent-labeled antibodies. After elution from the magnetic particles, quantification is performed using laser detection. ASO MSD assays are recommended for measuring ASO concentrations in tissue homogenates, such as biopsies from preclinical in vivo studies. On the other hand, SMC applications enable high-sensitivity detection of ASOs in biofluids such as cerebrospinal fluid (CSF) and plasma. This innovative SMC technology exhibits significantly increased sensitivity compared to MSD platform approaches and is suitable for clinical research applications. Figure 26. Successful detection of Malat-1 ASO by ASO immuno-based detection assay on the MSD platform. Concentration-dependent ASO MM2 and MM5 detection independent of matrix (liver) Good recovery (>4/6 data points within range of 80–120%) Successful detection of ASO MM2 and MM5 in in vivo samples (liver tissue from Malat1 in vivo study, 14 days after treatment, assay input 5 μg/μl protein) ASO Response ASO Recovery ASO Quantification in vivo Malat1 mRNA KD in vivo ASO MM5 assay ASO MM2 assay 3 6 Figure 27. Dynamic range and sensitivity of ASO detection on MSD versus SMCxPRO® platform. References 1 Thayer et al., POE Immunoassay: Plate based oligonucleotide electrochemiluminescent immunoassay for the quantifcation of nucleic acids in biological matrices. Scientific Reports | (2020) 10:10425. 100x fold increased sensitivity with SMCxPRO compared to MSD Good dilution linearity when ASO was spiked in human CSF, very good recovery of ASO down to 0.03 ng/mL. High sensitivity ASO detection by ASO SMC assay on SMCxPro patform Signal (SMCxPRO) Signal MSD) ASO spike in human CSF [ng/mL] 10 000 1 000 100 10 1000000 100000 10000 1000 100 10 MSD SMCxPRO 3 7 Semi-quantitative and Qualitative Methods High Content Quantitative Image Analysis Oligonucleotides can be visualized in any tissue of choice using the RNAscope™ technology. The RNAscopePlus™ assay, a highly-specific and sensitive in-situ hybridization assay, enables the detection of single oligonucleotide molecules. This assay is crucial for evaluating therapeutic ASO delivery, biodistribution and cellular uptake. We have implemented this method in-house and can combine it with immunohistochemical staining to localize ASOs in specific tissue regions, different cell types and even cell compartments within the same tissue section. By integrating in-situ hybridization with immuno-histochemical staining to visualize target proteins, this method also enables the correlation between ASO uptake and regulation of target proteins. In addition, the assay allows for the analysis of target proteins in PK/PD animal models. Oligonucleotides can be detected and quantified and their efficacy and duration on target proteins can also be analyzed. Quantification can be done on a single-cell level and multiplex-fluorescent image acquisition allows for up to 5 channels in parallel using a fast and automated Axioscan slide scanner. Automated image analysis is tailored to individual project needs and can measure parameters such as number of events, density, texture and more. Figure 28. Biodistribution of a therapeutic ASO in various regions of a mouse brain (cortex, hippocampus and cerebellum) as visualized by the RNAscopePlus™ assay. The ASO is specifically stained in yellow, with no background signal observed in untreated controls, indicating high specificity. The target protein, stained in red, is downregulated in all analyzed brain regions following ASO treatment. The ASO signal is differentially detectable in neurons, marked by NeuN and in non-neuronal cells. Additionally, the nuclear dye DAPI allows for the distinction between nuclear and cytoplasmic localization of the ASO. 3 8 High Content Imaging A typical image analysis workflow starts with the annotation of tissue-specific regions by experts trained in e.g., neuroanatomy (fig 29A). This way, subsequent evaluation of biodistribution can be stratified by brain areas like the hippocampus, cerebellum, striatum, or any other assessable anatomic brain region. In the next step, cells are typically identified by nuclear staining. Additional cellular or cell typespecific markers can be used to identify further cellular compartments, so that the localized staining signal can be evaluated (fig. 29B). The cellular localization of cell type-specific markers can be used to identify cellular populations e.g., neurons, astrocytes and microglia (fig 29B). When analyzing cellular staining, occasionally looking at only the signal intensity is not sufficient. Therefore, we routinely quantify additional signal features like morphology and texture. This allows us to identify suitable parameters for the identification of specific cell types and the evaluation of complex staining. In the case of an ASO-specific RNAscope, the staining is, for example, quantified by segmentation of the spot-like signal (see fig. 29B), after the application of a spot-texturefilter. If necessary or beneficial, images can also be analyzed using in-house developed Figure 29. A. Typical analysis workflows start with the annotation of tissue-specific regions, e.g., cortex, hippocampus and thalamus in brain sections. B. Immunofluorescent staining can be used to identify cellular subregions (e.g., nuclei, cytoplasm), or cell populations and to evaluate the signal-of-interest within these populations and subregions. C. Evaluation of single-cell data allows for the analysis and augmentation of results on a single-cell level. deep-learning classification algorithms. Employing artificial intelligence (AI) this way has proven useful when trying to identify brain region-specific cell populations. At the end of our analysis workflows, results will be either collected per tissue region or we will prepare single-cell results. The latter allows additional data exploration, evaluation of subtle effects within a given region and a more complex data augmentation (fig 29C). References 1 Thayer MB, Humphreys SC, Chung KS, Lade JM, Cook KD, Rock BM. POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices. Sci Rep. 2020;10(1). doi:10.1038/s41598-020-66829-6 A B C 3 9 in vivo ADME Characterization: in vitro Stability and Metabolic Profiling Several factors influence the in vivo pharmacokinetics and therapeutic efficacy of oligonucleotides, including their susceptibility to nuclease-mediated metabolism. We develop in vitro assays to investigate the stability of oligonucleotide drugs in various matrices and tissues during the lead optimization process. These incubations are performed in plasma or tissue homogenates from different species, with varying incubation times. The stability of each oligonucleotide is reported as the percentage of the intact molecule remaining at each time point compared to time zero and the half-life is calculated. To maximize the information obtained from in vitro stability assays, metabolite profiling can be conducted in parallel using high-resolution mass spectrometry platforms and dedicated software for data processing. Figure 30. in vitro stability assays and metabolic profiling are performed in parallel using high-resolution (HR) and low-resolution (LR) mass spectrometry (MS) platforms. In vitro stability Measured by incubation in homogenates Up to 8 timepoints over 72 hours Possibility to further process samples for early metabolities evaluation Stability in matrix of interest Half life (t1/2) = ln(2) / K Time (min) ln (% Parent Remaining) 0 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 50 100 150 200 250 300 350 400 rp1 rp2 4 0 in vivo ADME Characterization: Plasma Protein Binding Investigating drug-plasma protein binding is a crucial aspect of the drug development process, including for oligonucleotides, as it influences pharmacokinetics and biodistribution. Regulatory agencies such as the FDA and EMA emphasize the importance of plasma protein binding and advocate for the development of appropriate in vitro studies to examine this parameter. We have developed an ultrafiltration assay that integrates standard ultrafiltration methodology with LC-MS/MS analysis to specifically assess the plasma protein binding (PPB) of ASOs. This assay employs two known ASOs as positive controls for high and low binding. The PPB results obtained using our assay provide a reliable and generic approach for such investigations, yielding results consistent with reported data for the positive controls. Ultrafiltration (UF) - non-specific binding to membrane + relatively fast ultrafiltration procedure Key factors affecting target gene knock down: Favorable tissue distribution Potency Protein binding Protein Binding affects: toxicity, plasma half-life, tissue distribution/accumulation, cellular uptake, activity and renal excretion Protein Binding: is used to allometrically scale PK parameters Plasma protein binding (PBB) Measured by ultrafiltration Marketed ASO used as positive controls for low and high protein binding Plasma protein binding (% bound) Marketed compounds Volanesorsen 100 75 50 25 0 Eteplirsen Evotec Mouse Literature Mouse Other CRO mouse Evotec Human Literature Human Other CRO Human Figure 31. Plasma protein binding. 4 1 PK/PD Modeling Oligonucleotides have complex pharmacokinetic and pharmacodynamic relationships that need to be investigated and understood to allow translatability of a compound. Our integrated approach includes DMPK and PK/PD modeling functions from the start, allowing for the possibility to design in vivo studies in the spirit of the 3R’s, while maximizing the information obtained and possibly reducing the number of studies required. Expert modelers can implement PK and PK/PD models that can transition from discovery to development. Figure 32. Various strategies for PK and PK/PD modeling and human PK and dose prediction. PK Modeling NCA PK Comp. PK Semi-physiological Comp. PK Pop PK PK/PD Modeling Indirect link Effect Comp. PK/Emax DRT Human PK and Dose Prediction Allometry PK/PD nI i v ort Ex Vivo Response Dose Biophase Log PK Log PK=a·BWy Log BW 4 2 Figure 33. PK/PD and POP-PK/PD modeling approaches enabling the transition from preclinical species to humans. Constructing a multicompartmental pharmacokinetic (PK) model linked with a pharmacodynamic (PD) model is often necessary to accurately describe the PK/PD relationship of an oligonucleotide. In the example below, a Malat 1-targeting antisense oligonucleotide (ASO) has been modeled Mathematical PK/ PopPK modeling of data In vivo pharmacokinetic and pharmacodynamic data Mechanistic understanding PK/PD modeling leverages the temporal gaps between plasma and tissues exposure, as well as PD effect Understanding of PK/PD relationships for oligos Possibility to select optimum dose for efficacy and toxicological studies Bridging discovery with development activities Translation from preclinical species to human Support from preclinical to clinical studies based on plasma, liver and kidney exposures, as well as Malat 1 knockdown in the liver and kidney. The established PK/PD model can then be utilized to inform and optimize the design of repeat-dosing or efficacy studies, thereby increasing the likelihood of success (Figure 34). Mathematical modeling of PK/PD data Human PK and dose predictions 4 3 Figure 34. An example PK/PD modeling workflow using the Malat1 oligonucleotide (MM5). Mechanistic understanding Understanding PK/PD relationship of oligonucleotides in target tissue(s) is fundamental due to the different plasma/tissues kinetic PK (exposure by LC-MS) and PD (Malat 1 KD by gPCR) from a Malati oligonucleotide (MM5) were obtained after single subcutaneous (SC) administration to mice at dose levels of 1, 8 and 25 mg/kg PK/PD model design Malat 1 expression Malat 1 expression Plasma ka k12 k21 kelpk kelpl kelK kelk Other tissues Kidney Liver Dose PK/PD model application Pharmacokinetic Pharmacodynamic Liver PK/PD model construction Pharmacokinetic Pharmacodynamic Plasma Liver Liver Kidney Kidney 4 4 Pathology Pathology is an integral part of oligonucleotide development, offering crucial insights into drug safety and efficacy. This includes early pathology studies for target validation and lead optimization, in addition to preclinical toxicology screening and safety studies. Across these studies, rigorous microscopic examination and interpretation are required to detect any potential toxicity. To accompany early safety in vivo or latestage programs with developmental candidates, we have a panel of expert pathologists and toxicologists to help interpret in vivo safety. Below is an overview of our expertise and capabilities in oligonucleotide pathology. Microscopic Examination and Interpretation Pathologists: DVM, PhD and board-certified (ECVP or ACVP) Experience with different specialty areas, therapeutic modalities, RoA and animal models Time- and cost-effective peer reviews on glass slides or WSI Support to new drug modalities: Expertise with the safety of RNA therapeutics, incl. CNS ASO, gene and cell therapy-based products Neuropathology expertise to support direct delivery (intrathecal, intracerebroventricular, intraparenchymal) and in-dwelling catheter administration studies in multiple species Clinical Pathology Expertise Data interpretation for standard clinical pathology parameters Investigative biomarkers available (e.g. NFL), incl. validation and development Transmission Electron Microscopy: From sampling to ultrastructural interpretation Microscopic Imaging Services High-resolution slide scanning (Aperio GT450) Quantitative image analysis using open source or customized imaging software (Visiopharm), incl. AI-assisted 4 5 Figure 35. GLP-compliant histochemical and immunohistochemical techniques. GFAP (astrocytes) IBA-1 (microglia) Figure 36. Histological examination of the GFAP stain and IBA-1 biomarker in different CNS cells. Neuroanatomy-based matrix-guided trimming protocol Matrix channels selected Standardized trimming protocols (e.g. NTP 7 levels in rodents) Target brain levels requested Blk 3A 1 Blk 3B 2 3 Blk 3C 4 5 Blk 3D 6 7 4 6 Preclinical Development The preclinical development phase is aimed to support clinical trials and to get regulatory approval (i.e. IND/CTA enabling) for use in patients. The preclinical safety assessment framework spans from late lead optimization to the first administration to humans, as highlighted below: We have extensive capabilities to ensure an end-to-end oligonucleotide R&D continuum, with strong synergies across discovery being translated to the full development paradigm. This allows a fast and patient-oriented process to IND/CTA and clinical phases. From the regulatory point of view, oligonucleotides represent a challenging field as they share properties with both chemical and biological pharmaceuticals. Considering the already marketed ONDs, most companies have adopted the two species small molecule approach, as outlined by the ICH M3(R2) guideline. Figure 37. The preclinical IND/CTA enabling phase, spanning from late lead optimization to Phase 1 clinical trials. Species Selection According to the international guidelines, cross activity and full pharmacologic action should be reproducible in at least one of the two animal species adopted. The non-human primate (NHP) is by far the most common non-rodent species used for both ASO and siRNA candidates. This is mainly due to the highest likelihood of on-target sequence-dependent crossover and a robust historical background record. Rodent species used may vary from rat or mouse and include the eventual use of transgenic or immunosuppressed animals. If a human active candidate with limited cross-species activity needs to be developed (even if NHP are considered), a surrogate molecule with proven activity in the chosen rodent species can be included. This should be tested in parallel to the clinical candidate and will enable adequate assessment of the on-target toxicity. Drug Discovery Phase I Phase II Phase III Approval Market Clinical Enabling Lead Optimization 4 7 We have the capabilities to design and develop a surrogate ASO to match the same design and chemistry as the clinical candidate. These have a good general safety profile, to allow the robust assessment and documentation of any potential on-target toxicities. Considering that developmental and reproductive toxicology studies (DART) and carcinogenicity studies are run in rodents and rabbits, surrogate ASOs must also be used for OND that are only active in NHP. Our Verona site has GLP capabilities and can offer AAALAC-accredited facilities for preclinical integrated studies with rodents, dogs and NHP: Figure 38. AAALAC-accredited animal facilities for preclinical integrated studies. Rodents – PK; PK/PD; Disease models; Toxicology; Safety Pharm – Males and females – 37 rooms, up to 7-8K – Capacity: 15-20 28d studies/year – 6,000 sqm – Facilities certified as Bio Safety Level 2 (BSL2) area Dogs (Marshall Beagles – US and France) – General Toxicology (males and females) – PK (males), CV (males) – 6 kennels – 161 pens – Capacity: 10 28d studies/year NHP (Cynomolgus monkeys/ Macaca Fascicularis – Mauritius and Asia) – General Toxicology (males and females) – PK (males), CV (males) – 6 housing rooms 70-100 animals – Capacity: 6 28d studies/year Rabbits – PK, Early Tox (capacity limitations), investigative studies (males and females) 16 animals – exploring capacity expansion 4 8 Toxicological Profile In general, oligonucleotide-based pharmaceuticals display a common toxicological profile: Adverse effects on the liver and kidneys Clinical pathology alterations: Hematological changes, prolongation of the coagulation time and activation of the complement system Immunostimulation It is possible to fully characterize and monitor these effects in the preclinical studies to support human administrations. Figure 39. An overview of the preclinical investigations for OND toxicity. OND toxicity Predictive assays In vitro/ in vivo Off-target effects In silico evaluation/in vitro validation In silico and in vitro Immunostimulatory effects – Quantification of cytokines/chemokines release in human PBMC or WBA by ELISA In vitro – Quantification of cytokines as well as CCL22 mRNA levels in BJAB cells by qRT-PCR Toxicities in high exposure organs – Cytotoxicity by caspase assay In vitro Predictive assays for hepatotoxicity: – Quantification of LDH and ATP in primary hepatocytes In vitro – Caspase assay in transfected mouse 3T3 fibroblasts or human HepG2 cells In vitro – Evaluation of liver enzymes in mice/NHP In vitro Predictive assays for nephrotoxicity: – Quantification of EGF in human kidney tubule epithelial cells In vitro – Quantification of kidney injury biomarkers using chip-cultured HRPTEC In vitro – Quantification of urinary biomarkers (eg, β2-microglobulin and KIM-1) by ELISA In vivo Thrombocytopenia – Evaluation of platelet activation in human or HP platelet-rich plasma or whole blood by flow cytometry (activation of CD62P and PAC-1) In vivo and in vitro Inhibition of coagulation – Quantification of PT and aPTT in vitro in human/mouse/NP citrated plasma In vitro Complement activation – Quantification of split products of the APC (C3a, Bb, and Ca) in vitro in human/NHP/mouse serum In vitro CNS-specific toxicities – Prediction of neurotoxicity from sequence features In silico – Quantification of spontaneous calcium oscillations in primary cortical neuronal cultures In vitro 4 9 Preclinical Toxicology Study Design In the IND/CTA enabling phase, both study duration and route of administration should match those intended to be adopted in clinical trials. The following administration routes are feasible and can be adopted in our GLP studies: Intravenous or subcutaneous Intrathecal or intracerebroventricular (ICV) targeting CNS Intra-organ specific delivery (i.e. surgery-based techniques) Surgical Capabilities Consolidated capabilities of performing tailored surgical procedures in rodents, to ensure out-of-standard route of administrations. Some examples: Intracerebroventricular Intrathecal Intra-articular Intra-cochlea Intra-myocardium Intra-pancreatic Specific formulations aimed to improve absorption can be considered (i.e. LNP coated OND) and a full toxicological and immunotoxicological evaluation on the combination can be provided. The duration of the preclinical GLP studies ranges from 1 to 3 months for the IND/CTA enabling phase, up to chronic studies of 6 and 9 months for rodents and non-rodents, respectively. Doses are not expected to be high: They are normally expected within the 20–80 mg/kg range. We normally design preclinical toxicology studies as fully integrated studies, including the following pharmacology and efficacy endpoints: Toxicology endpoints: Clinical pathology, histopathology, specific biomarkers, immunotoxicology/ADA Biodistribution assessment: Plasma and tissue sampling for OND exposure and persistence assessments (with time-course capabilities) PK/PD and efficacy endpoints (i.e. gene and protein expression, immunogenicity) 5 0 Safety Pharmacology As for small molecules, in vivo safety pharmacology studies are required to rule out adverse functional effects on key organs such as the CNS, cardiovascular and respiratory systems. In vivo models (GLP/Non-GLP) CV: in vivo telemetry (ALL species) – Combined Neuro-Cardio dog model (DOG) CNS: Irwin/neurobehavioural FOB testing (ALL species) Respiratory function – WBP (rat) Figure 41. Safety pharmacology study to assess the potential impact of a drug candidate on the electrical activity of the heart. P wave Q S R T wave QT interval Electro-Cardio-Gram K+ channel block ( AP Duration) I Kr (hERG) I to (K v4.3, Kv1.4) I Ks (KCNQ1) I K1 (Kir2.1) I h (HCN4) I Ca (Ca v1.2) I Na (Na v1.5) Cardiac Action Potential Figure 40. GLP and non-GLP in vivo models for cardiovascular, CNS and respiratory systems. 5 1 Genotoxicology Profile Despite the general lack of positive results in regulatory genotoxicity studies, several health authorities still request in vitro and in vivo genotoxicity assessments. Therefore, evaluation strategies to test the genotoxicity of “non-natural” oligonucleoRegulatory Framework and Considerations The regulatory framework and related guidelines that are generally adopted are reported below: No specific guideline is currently available for ONDs ICH3 (M2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (International Council for Harmonization) ICH S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals OECD 1&19: OECD Series on Principles of Good Laboratory Practice (GLP) and Test Item Characterization according to The Organization for Economic Co-operation and Development FDA Guidance for Industry: Gene Therapy Clinical Trials – Observing Subjects for Delayed Adverse Events; November 2006 tides should preferably consider mammalian-based in vitro tests in addition to the in vivo rodent-based evaluations, as follows: In vitro mouse lymphoma assay In vitro micronucleus test Rodent micronucleus test To design the preclinical development strategy, a tailored risk assessment needs to be undertaken on a case-by-case basis, depending on the indications and target patient population. In that regard, several of the currently approved oligonucleotide products (e.g., eteplirsen, mipomersen, inotersen, volanesorsen) are aimed to treat rare, often genetic diseases for which no alternative therapy is available. In such cases, the presence of minimal safety concerns was considered acceptable. However, new therapeutic indications aimed to treat significantly larger populations with more common diseases (e.g., cholesterol-lowering siRNA), would require an improved risk-benefit assessment. 5 2 Quality and Compliance Our Verona site is GLP and GCP certified, with strong and consolidated experience in quality and compliance. Flexible and tailored study designs are a must have. We can ensure this under the highest quality standards. Integrated and complex preclinical studies are made possible by combining toxicology and biodistribution GLP evaluations – which are always required to support human administration – with non-GLP efficacy and PK/PD assessment in the same study. These combined studies allow a reduced and more ethical use of animals. In compliance with: OECD Principles on Good Laboratory Practice 1997 ENV/MC/CHEM(98)17 The GLP testing facility is regularly inspected by the MoH GLP Monitoring Unit every 2 years Figure 42. Over 35 years of GLP and GCP compliance at the Evotec Verona site. (as GSK) Oct 1987 Italian Ministry of Health May 1991 Italian Ministry of Health Jul 1993 FDA Oct 1998 Italian Ministry of Health Feb 2001 Italian Ministry of Health Sep 2005 Italian Ministry of Health Oct 2008 Italian Ministry of Health Aptuit/Evotec Sep 2011 Italian Ministry of Health Feb 2013 Italian Ministry of Health Mar 2015 Italian Ministry of Health May 2017 Italian Ministry of Health July 2019 Italian Ministry of Health Oct 2023 Italian Ministry of Health 5 3 Regulatory Support Bringing novel oligonucleotides to market presents unique scientific and regulatory challenges across the pipeline, including API and impurity characterization. Our Global Regulatory Affairs (GRA) experts will strategically guide you through the hurdles and challenges of an uncommon drug development including support to orphan drug designation and patient selection, as appropriate. In addition, our GRA department, formed by a team of nonclinical, clinical and CMC experts is dedicated to assisting with the preparation and submission of regulatory documents, including INDs and CTAs, to the relevant authorities and to support the strategic preparation of meetings with the health authorities. The GRA group can support in the following activities: Global Regulatory Strategy (GRS), including the Target Product Profile (TPP) Support meeting with EMA such as Innovation Task Force meeting and CHMP Scientific advice or Local national scientific advice Support the formal meetings with FDA, i.e. INTERACT and pre-IND meeting US and EMA orphan drug designation US-IND preparation, finalization and submission to FDA through a validated EVT RIM VAULT system CTA preparation, finalization and submission through the central CTIS portal A critical element for a successful regulatory strategy is the provision of a clear product path to registration in the target disease and patient population (therapeutic indication) (i.e. accelerated programs, early interaction with agencies, orphan status, R&D incentives). This is achieved by analysis of the guidelines available, the precedents, or potential program-specific agreements with health authorities. In some cases, it might require additional negotiation with health authorities, for example when no guidelines, no recent drug approvals, or no validated clinical endpoints are available. The regulatory strategy gives clear guidance to the drug development participating functional teams to ensure critical strategic regulatory elements are considered and embedded throughout the product development. As medicinal products continue to evolve in complexity (such as ASOs), a robust and comprehensive regulatory strategy document is at an all-time advantage. The Target Product Profile (TPP) is included in the regulatory strategy. To cite the WHO definition, “a TPP outlines the desired ‘profile’ or characteristics of a target product that is aimed at a particular disease or diseases. TPPs state intended use, target populations and other desired attributes of products, including safety and efficacy-related characteristics. Such profiles can guide product research and development (R&D)”. Our GRA team will collaborate strictly with the ASO developers to support the definition of a successful TPP. 5 4 Therapeutic Area Expertise With complexity and competition rising in drug R&D, success requires both technological innovation and in-depth therapeutic expertise. Our extensive expertise across a wide range of disease areas, including CNS disorders, oncology, metabolic disorders and rare diseases to name a few allows us to bring projects forward with a higher rate of success. Efficient Problem Solving: Disease experts can anticipate potential challenges and address them proactively, reducing delays and improving the overall efficiency of the drug development process Regulatory Insight: Knowledge of diseasespecific regulatory requirements helps in navigating the complex regulatory landscape, ensuring compliance and facilitating smoother transitions from preclinical to clinical phases Enhanced Data Interpretation: Experts can provide deeper insights into the data collected during preclinical studies, leading to more accurate interpretations and better decision making in subsequent phases Innovative Approaches: Disease expertise fosters innovation by applying the latest scientific advancements and methodologies to the discovery and preclinical stages, potentially leading to breakthrough therapies These benefits highlight the importance of disease expertise in enhancing the quality and success rate of drug discovery and preclinical studies at Evotec. Figure 43. Broad therapeutic area expertise covering a wide range of diseases. This expertise offers several key benefits: Targeted Drug Development: Disease experts can identify and validate drug targets more effectively, ensuring that the compounds being developed are relevant and have a higher chance of success in treating the disease Optimized Study Design: Expertise in specific diseases allows for the design of preclinical studies that closely mimic the conditions and variables expected in human trials. This includes selecting appropriate models and endpoints to measure efficacy and safety CNS diseases Muscle diseases Metabolic diseases Liver diseases Oncology Autoimmune disease Virology Respiratory diseases Aging Rare diseases Kidney diseases Fibrosis Immunooncology Inflammation AMR TB Women’s Health 5 5 Thank you for reading our oligonucleotide ebook. We hope that you have found what you’ve been looking for, but if not, please get in touch. We are a team of curious and passionate scientists, and we love a challenge. The way we work is flexible. We will meet with you to get an idea of what you have in mind and then we will assemble a team to discuss what we would propose to do to tackle your specific request/problem/idea … its free! After discussing your project with you, we will draw up a proposal of work, with timelines and costs for you to get a better idea of how to progress. You can pick and choose, you can argue, or you can fall in love with our ideas. Either way, you decide if you want to progress past this proposal stage to a work order. When a project starts we will share the data obtained, our analysis and interpretation and we will recommend the next steps and then we will decide together with you how to progress. In this process, we are your team. We pull in our scientific experts as they are needed on the project, in silico, in vivo, DPMK, omics, biophysics, bioanalytics etc.. A dedicated project manager will track the spending for you monthly. You pay only for the work that together we agree will be completed. This allows for your project to be driven by data, to follow the biology and adapt to the needs and challenges of each project as they arise. If you’re interested to learn more, please reach out. We would love to discuss your project with you and what we can do to work together. Closing Remarks Evotec SE Manfred Eigen Campus Essener Bogen 7 22419 Hamburg (Germany) info@evotec.com | www.evotec.com Learn more: