Key Themes of the Event
Both live and on-demand, 175+ presentations and discussions explored a diverse range of topics across the spectrum of oligonucleotide & peptide therapeutics.
Biomedical researcher and writer Dr. Catarina Carrao reviews the key themes of the event, addressed through popular keynotes and panel discussions.
Key Themes of the Week - Executive Summary
Oligonucleotide CMC Strategies for Quality Control - Panel Discussion
The dialogue started with the importance of sequence confirmation for oligonucleotides, which continues to be a debated issue. In the point of view of Dr. Rene Thürmer, Deputy Head at BfArM Federal Inst for Drugs & Med Device, sequencing should always be part of the drug substance specification in identity testing; and, a tip given by him of a good insight on this topic is the recent publication by Capaldi and colleagues on this regard (Nucleic Acid Therapeutics, August 2020).
Dr. Claus Rentel, Executive Director, Analytical Development/QC at Ionis Pharmaceuticals, Inc. asked Dr. Thürmer about the possibility to use Next Generation Sequencing to validate oligonucleotide sequencing, which he agreed to be a possibility; even though, in relation to MS/MS-sequencing, Dr. Rentel was critic of the fact that it’s difficult to fully sequence the oligo with this technique.
When questioned about low-level impurities, Dr. Thürmer remitted to his talk that justifications based on science and ICH Q11 should always be provided; with a comprehensive data set, characterization, suitable and validated analytical methods (which are usually not required for small molecule starting materials), description of synthesis and tight specifications (i.e., justification for each attribute should guarantee consistent quality). Also, he stretched that there needs to be a high number batch of analysis to demonstrate consistency, with an elucidation and criticality assessment of the impurity profile; and genotoxic impurity assessment in line with ICH M7 requirement.
Criticality of starting materials and impurities was a point raised by Dr. Rentel, which was also supported by Dr. Thürmer, emphasizing how significant this aspect of the regulatory file is for the marketing approval.
"Sequencing should always be part of the drug substance specification in identity testing"
Targeted Delivery of Oligonucleotides - Panel Discussion
Dr. Punit Seth, Vice President, Medicinal Chemistry at Ionis Pharmaceuticals, started by answering a question about target delivery in the muscle. His recent research has shown that attaching palmitate, tocopherol or cholesterol to phosphorothioate (PS) antisense oligonucleotides (ASOs) can influence their activity and distribution in muscle tissues, increasing the possibilities of breaching extra-hepatic delivery of oligonucleotides.
The next question to the panel related to tumor vasculature via integrin targeting, to which Dr. Seth replied that they have tried RGD peptide (Arginylglycylaspartic acid) and were disappointed with the results.
Dr. Muthiah Manoharan, Senior Vice President of Drug Discovery at Alnylam, further replied that integrins can be the right target, even though research is incomplete. He added that previous studies together with Prof. Rudy Julyano actually showed a variety of approaches to link oligonucleotides to integrins with high specificity and affinity, including direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers.
Next, the audience asked what were the receptors responsible for the FALCON (Fatty Acid Ligand Conjugated OligoNucleotide) conjugates, to which Dr. Charles Allerson, Vice President of Chemistry & Drug Development at DTx Pharma, replied that this specific platform offers receptor-mediated uptake through proprietary fatty acids, even though they are still not sure of which specific receptors are responsible for delivery.
As moderator, Dr. Manoharan said the audience was interested in antibody oligo-conjugates (AOCs), to which Dr. Seth responded that there is already proof-of-concept publications for this new emerging therapeutic application; and, in his opinion, the exploitation of protein binding specificity of antibodies to deliver ASOs for the silencing of cell type-specific genes, will continue to grow.
Afterwards, the enthusiastic panel members took questions from each other, with Dr. Allerson asking Dr. Seth about Ionis’ oral delivery of oligo compounds, to which he replied that they used a permeation enhancer to open the tight junctions and cellular transporters, work that has been previously published by Ionis. Dr. Seth was then interested in the lipid transporters studied at DTx, to which Dr. Allerson replied that, even though they haven’t found specific transporters yet, they are able to compete the uptake with fatty acids in an active mechanism.
Peptides Past, Present and Future: A Peptide Drug Hunter Odyssey - Keynote Presentation and Live Q&A
Dr. Tomi K. Sawyer, Chief Drug Hunter and President at Maestro Therapeutics, gave an exciting keynote presentation about the odyssey of peptide drug discovery, and how it has evolved from highly focused efforts on specific receptors and proteases, to a plethora of targets bridging receptors to enzymes and protein–protein interactions. As he explained, penetrating into cells to modulate intracellular targets is no longer a challenge, opening the “druggable” target space.
Dr. Sawyer explained that over recent years, peptide macrocyclization has seen a rebirth as a key driving force of the third wave of peptide drug discovery. Leveraging both structure-based design and super-diverse library screening of synthetic peptides has accelerated the generation, optimization and development of these types of peptide drugs.
Macrocyclic peptide natural products (e.g., cyclosporine A or CsA), designed macrocyclic peptidomimetic inhibitors of proteases (e.g., hepatitis C virus (HCV) protease inhibitor), designed stapled α-helical peptides (e.g., dual MDM2/X antagonist), CsA-inspired mRNA display library-generated macrocyclic peptides (e.g., SIRT2 inhibitor), CsA-inspired DNA encoded library-generated macrocyclic peptides (e.g., XIAP antagonist) and cell-penetrating peptide (CPP) hybridized synthetic library-generated macrocyclic peptides (e.g., KRAS antagonist) are providing novel chemotypes with unique biophysical and pharmacological properties to tackle the so-called “undruggable” targets.
There is significant progress in the study of stapled α-helical peptides, and the application of varying synthetic chemistries (e.g., ring-closing metathesis, azide–alkyne cycloaddition and thioether). The highly potent and in vivo effective dual MDM2 and MDMX antagonist ATSP-7041 (Aileron Therapeutics) has become a benchmark stapled peptide, due to its intrinsic biophysical properties (e.g., amphipathicity, solubility, cell permeability and metabolic stability); and, has successfully progressed into the clinic.
Using the ATSP-7041 has a model system, with a combination of computational and innovative experimental and predictive screening tools, has allowed the exploration of the intracellular target space with a collection of resources of structurally diverse macrocyclic peptides along with design rules that advance the peptide-drug world. The third wave of peptide discovery is rising and it will be huge.
"Peptide macrocyclization has seen a rebirth as a key driving force of the third wave of peptide drug discovery"
Personalized Peptide Vaccines - Panel Discussion
Moderator Dr. Trishul Shah, Head of Sales, North America at PolyPeptide Group, Director Business Development, started the discussion by asking Dr. Catherine Wu, Professor of Medicine at Dana-Farber Cancer Institute and Harvard Medical School, about the car analogy in her path towards the discovery of personalized peptide vaccines. Dr. Wu responded from an oncologist point of view, that the knowledge about checkpoint blockade is comparable to “taking the foot out of the brake pedal”, and has allowed “opening the door” of the tumour and “speeding up the way” for an efficient patient therapy.
Next, Dr. DeOliveira, Sr. Director, Peptide Development, Tech, Ops. & Manufacturing at Genocea Biosciences, talked about the proprietary ATLAS platform that enables an optimization of neoantigens for inclusion in immunotherapies, by excluding specific inhibitory antigens that suppress the patient immune system, the so-called Inhibigens. In his opinion this will be a “game-changer” in the field of personalized peptide vaccines, by allowing a blockage of the internal growth signals specific of the cancer cells.
"Knowledge about checkpoint blockade is comparable to “taking the foot out of the brake pedal”, and has allowed “opening the door” of the tumour and “speeding up the way” for an efficient patient therapy"
When questioned whether Inhibigens could be a neoantigen for a different patient, Dr. DeOliveira replied that their research shows that neoantigens and Inhibigens tend to have the same features across patient profiles.
Asked how the decision is made between which patient takes Genocea’s GEN-009 or GEN-011 personalized vaccines, Dr. DeOliveira replied that even though preliminary, GEN-009 is meant for a patient that still has a viable immune system that drives the anti-tumour response.
On the contrary, since the GEN-011 is an ACT or 'adoptive T cell therapy', it involves the isolation and expansion of tumor- or neoantigen-specific T cells to create the therapeutic; as such, it is meant for a situation where the patient is already immunocompromised and needs a full battalion of engineered T-cells to fight the tumor.
Dr. Annie De Groot, Founder, CEO and CSO at Epivax, Inc., joined the panel discussion, and asked whether the oncologists were concerned with impurities in the starting materials for the personalized vaccines.
Dr. Wu replied that they usually “take a really good look” at the traces of the manufacturers, to make sure of the specificity of the product. They then complete this analysis in the lab, with a sound track of product testing (e.g., Mutant vs Wild Type) to be certain of what they will be injecting into the patient and possible side effects.
Dr. Jesse Dong, Vice President, Peptide Chemistry at BioNTech US, was asked about the induction of neoantigen-specific immunity by NEO-PV-01 with adjuvant anti-PD-1. He replied that they have seen epitope spreading of the immune response to neoantigens that were not included in the vaccine in 15 out of 22 patients, which further shows that the vaccine induced a broadened immune response and had positive effects on survival. Dr. Dong further emphasized that these results, together with the results from Dr.Wu that show that the personalized vaccine alone has epitope spreading effects (with no adjuvant added), are very promising for the future of personalized cancer treatment. In his and Dr. Wu’s opinion, this is a very exciting outlook for the field.
Novel Technologies for the Delivery of Macromolecular Therapeutics - Panel Discussion
The moderator of this engaging panel session, Dr. Stephen Spagnol, Senior Scientist, Large Molecule Drug Product Development at Johnson & Johnson, began by asking what the panelists’ thoughts are on the major challenges for the delivery of macromolecules as therapeutics.
Dr. Krishanu Saha, Associate Professor at University of Wisconsin-Madison, explained how, in their research, they have tried traditional agents for nonviral polymeric delivery of protein-based CRISPR systems, but ended up using the protein-RNA complex in itself, taking advantage of the cell’s natural mechanisms to deliver the payload to the nucleus. In his opinion, in the genome-editing space, protein-RNA therapeutics are very promising because, for CRISPR systems, there is only the need for a short half-life of activity, since the modifications are stable over time. Longer exposure could conduct to enhanced cell effects which are not desirable.
Dr. Vadim Dudkin, Senior Director & Head, RNA & Targeted Therapeutics, Discovery Sciences at Janssen R&D, added that his research has focused not just on surface binding to the target, but in increasing the capacity for internalization and kinetics, as a way to rise the dosage of the payload to the targeted cell and its native biodistribution properties. In his opinion, understanding of intracellular trafficking and endosomal escape, both short and long-term, is of the utmost importance for an effective siRNA.
Dr. Craig Duvall, Cornelius Vanderbilt Professor of Biomedical Engineering at Vanderbilt University, then talked about how much time of his research has been dedicated to screening methods for the most endosomal-escape effective ligands. They have designed two genetically encoded split-luciferase turn-on reporter assays that can be measured rapidly in well plates on live cells using a luminometer: G8C2 system and G8G8 system.
Next, Dr. Spagnol asked Dr. Jessica Rouge, Assistant Professor of Chemistry at University of Connecticut, about the advantages of enzymatic-responsive materials, a focus of her research work. Dr. Rouge said their research seeks to repurpose enzymes to recognize peptide-modified oligonucleotide substrates for the sequence-specific assembly of RNA/DNA at the surface of micelle-like nanomaterials. The idea is to protect the native function, using mild and biocompatible attachment-cleavage chemistries naturally used by enzymes or exosomes.
Dr. Sriram Sathy, Vice President, Biology and Translational Medicine at Codiak Biosciences, “took the bait” and emphasised how Codiak has developed the engEx Platform, which engineers’ exosomes with distinct properties and direct tropism, so they can reach specific target cells and load them with various types of therapeutic molecules. They have discovered the surface glycoprotein PTGFRN as an optimal protein scaffold, that is amenable to genetic fusion with a broad class of therapeutic proteins.
Messenger RNA: Design and Manufacturing of a New Therapeutic Drug Class - On-demand Presentation
Dr. Anton McCaffrey, Senior Director of Emerging Science & Innovation at TriLink BioTechnologies Inc., explained that TriLink’s proprietary co-transcriptional capping procedure generates CleanCap in an efficient reaction, producing an extremely low-immunogenic cap with a natural structure that promotes an efficient translation.
Pseudouridine-5'-triphosphate (Pseudo-UTP) is used to impart desirable mRNA characteristics such as increased nuclease stability, increased translation or altered interaction of innate immune receptors with in vitro transcribed RNA. Pseudo-UTP, along with 5-methylcytidine-5'-triphosphate (5-methyl-CTP) have shown innate immune suppression in culture and in vivo, while enhancing translation. Dr. McCaffrey emphasized that TriLink is a leading manufacturer of Pseudo-UTP and chosen by many researchers in the field.
Next, Dr. McCaffrey elucidated that a variety of mRNA purification options are also available at TriLink, including silica gel purification, liquid chromatography isolation (LC-Isolation), and Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). According to his words, for some applications, LC-Isolation may be enough; although, the extra RP-HLC purification step is able to further reduce interferon-induction in THP-1 cells in many cases, increasing the efficiency of the mRNA.
Dr. McCaffrey then talked about self-amplifying RNAs (saRNA) and how this is an exciting new platform for rapid development of potent vaccines; where TriLink and Imperial College of London are collaborating to produce a saRNA vaccine encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP).
Dr. McCaffrey ended his talk by saying that TriLink takes pride in providing GMP mRNA manufacturing suites for all clinical needs, and a collection of analytical services that allow a thorough characterization of therapeutic mRNA.
Streamlining the Transition from Discovery to Manufacturing - Panel Discussion
Dr. Angelika Fretzen, Technology Translation Director at Harvard’s Wyss Institute for Biologically Inspired Engineering, was the moderator of the panel session, and asked how important are the milestones before researchers can go into manufacturing.
Dr. Weston Daniel, Vice President of Translational Research at Exicure Inc., said the first thing to think about is the basic: know your drug-product; what does the research group/start-up want to achieve with manufacture runs (e.g., supporting toxicology, clinical trials, etc.), and how much material needs to be made in the long-run? The advice is that, even though expensive, hiring a consultant will probably pay off, because many issues in Regulation, Quality Control and Manufacturing are not easy to perceive at first.
"Even though expensive, hiring a consultant will probably pay off, because many issues in Regulation, Quality Control and Manufacturing are not easy to perceive at first."
Dr. Andrew Latham Director, Business Development & Licensing at Merck was then asked how he looks at the licensing discussions, to which he replied that having an experienced CRO has many advantages when negotiating with a large company like Merck; also, a second source of supply is always important. Dr. Fretzen replied that in her in experience, sometimes a smaller COO and a small biotech company have a better alignment. Dr. Daniel agreed and emphasized that the right sized partner is very important for a successful move.
All panel members agreed that having a strong relationship between partners from the beginning onwards is as an important aspect for a successful endeavour. This relationship can take quite some time to grow, and timing needs to be right for both associates. As such, it’s important to focus on defining the product-drug critical attributes; make sure that “no skeletons are hiding in the closet” of stakeholders that could hunt the pipeline in the future; and, ensure that a trust bond between partners is built upon. Also, an alignment of interests and a global momentum goes a long way, and ensures that diligence talks move speedily and effectively.
Top Keynote Presentation: Tuning siRNA Potency, Specificity and Distribution through Chemistry
Prof. Anastasia Khvorova, Professor, University of Massachusetts Medical School
Tuning siRNA Potency, Specificity and Distribution through Chemistry
In one of the week's most popular keynote presentations, Prof. Anastasia Khvorova, Professor at University of Massachusetts Medical School, explained the methods of optimizing siRNAs within the chemistry process. Here we explore the highlights.
According to Prof. Khvorova, optimizing siRNAs for delivery is “like playing with Lego”, with many variables involved; and, the key to success is a combination of a well-designed siRNA, an optimized linker and a lipid conjugate (e.g. Docosanoic Acid, DCA).
Prof. Khvorova’s research group is trying to understand if the structure of the conjugate is the main factor that drives siRNA delivery; and, howthe siRNA structural conformation impacts accumulation in the tissue. Conjugation of siRNAs to lipophilic groups support an efficient cellular uptake, with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization.
Furthermore, these cholesterol-modified siRNAs (sd-rxRNA) are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) trafficking pathways in a two-step process: rapid membrane association, followed by internalization through a selective, saturable subset of the endocytic processes.
Prof. Khvorova then showed that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs (hsiRNAs) support a rapid cellular uptake and efficient mRNA silencing. Her work suggests that siRNA tissue accumulation does not fully define efficacy, and that the impact of siRNA chemical structure on activity is driven by intracellular re-distribution and endosomal escape. In her words, fine-tuning siRNA chemical structure for optimal extrahepatic efficacy is a critical next step for the progression of therapeutic RNAi applications beyond the liver.
In the following Q&A, moderated by Dr. Sanghvi, from Rasayan Inc., the first question related to the specific receptors involved in the molecular mechanism uptake of siRNAs. Prof. Khvorova responded that there could be an overlap of cumulative non-specificity; and, although, in some cases, like in the lung cells, there could be some receptor-specificity, the experimental work is very difficult to conduct.
In relation to the effect of tail-length on PS modifications relative to accumulation and efficacy with different conjugate platforms, Prof. Khvorova answered that her group saw similar results as those seen using the GalNAc platform. They see a measurable increase in accumulation, but it does not correlate with enhancement efficacy. Prof. Khvorova added that this phenomenon of phosphatides and single-stranded phosphatides having an extra push for cell internalization was not necessarily conjugate-dependent.
"Fine-tuning siRNA chemical structure for optimal extrahepatic efficacy is a critical next step for the progression of therapeutic RNAi applications beyond the liver."
Prof. Anastasia Khvorova, Professor at University of Massachusetts Medical School
The following question elaborated on the major limits for cholesterol in extrahepatic delivery and toxicity, to which she responded that between cholesterol and DCA, the latter gives a better accumulation and efficacy to the majority of tissues, with less risk. But of course, in her opinion, cholesterol conjugates can be very efficient in research and mouse models; and might be of use for local delivery of therapeutics.
Next, the audience asked if Prof. Khvorova had looked at the spectrum of plasma protein binding for different conjugates. Even though the answer was yes; unfortunately, the results revealed that a lot of the in vitro binding affinities did not hold true when translated to an in vivo environment. As such, this is a work in progress in her lab, where novel methodologies are needed to progress.
Prof. Khvorova was asked how to design the best linker for the most efficient escape from the endosome, to which she replied that the one they use the most is DTDT. “No cost, no tox, no nothing” were her actual words in relation to this linker.
Since the difference between in vitro and in vivo experimentation are quite different in terms of toxicity, the linker lipid conjugate is very important for endosomal escape and a driver for toxic reactions, in her opinion. She further explained that cleavable linkers have to be relatively stable in a series of time periods, so they can reach the target systemically after subcutaneous injection.
The next question was whether any high order structure, such as micelles, have been observed. Prof. Khvorova responded that micelles were detected with trimeric structures; but, in her opinion, the formation of such structure is contra-productive because it accumulates in the sub-cutaneous injection site, without further delivery to the target zone. Besides, when aiming to deliver to muscle tissues, for example, the compound actually needs to transfect the vascular epithelium. As such, in her view, the best compound is the most natural compound, one that is able to be transported through the cell internal mechanisms.
Hot Topic Keynote Presentation: mRNA as Medicine
Dr. Melissa Moore, Chief Scientific Officer, Moderna Therapeutics, Inc.
Hot Topic Keynote Presentation: mRNA as Medicine
One of the most exciting keynotes of the week was delivered by Dr. Melissa Moore, Chief Scientific Officer, Moderna Therapeutics, Inc., revealing the latest uses of messenger RNA in drug and vaccine development.
Dr. Melissa Moore, Chief Scientific Officer, Moderna Therapeutics, Inc., started her presentation by saying that, at Moderna, it all starts with an idea for a new therapeutic protein and its unique aminoacid sequence.
This is then back-translated using Moderna’s proprietary computer algorithm, which has been refined over the years to pick the best mRNA sequence for delivery.
For mRNA vaccine development, four essential steps are needed: (1) efficient methods to make large quantities of individual mRNAs at high purity; (2) ability to avoid the innate immune sensors that defend against RNA viruses; (3) efficient delivery methods; and (4) knowledge of how to engineer the best mRNA sequence for a particular purpose.
To achieve the first point, the Clinical train at Moderna manufactures larger batches of 5-75g+ mRNAs. These are currently being scaled up with manufacturing collaborators, with the objective of making 10x Kilograms of mRNAs in the near future.
Dr. Moore then explained that, to achieve the second point of avoiding the innate immune sensors, two type of receptors need to be eluded: Toll-like receptors (TLR), and RIG-I/MDA-5-like receptors. To avoid TLRs, Moderna mRNAs are engineered to replace all U’s with Pseudiuridines, which are natural occurring nucleotides that exist in ribosomal RNA and tRNA in our bodies and do not affect base-pairing.
To escape the RIG-I/MDA-5-like receptors, Moderna avoids the creation of double-stranded RNA (dsRNA) with state-of-the-art HPLC purification. This technique has just been published in Science Advances (June 2020). At this point, Moderna’s researchers have decided to go a step further, and reengineered T7 RNA polymerase not to produce dsRNA at all. A publication will soon come out, according to Dr. Moore; but for now, a recording of the last Annual Science Day presentation is available at Moderna’s website (June 2nd, 2020).
For the third step of an efficient mRNA delivery, most Routes of Administration (ROAs) require encapsulation with Lipid nanoparticles (LNPs, 80-100nm). At Moderna, each encased particle contains 2-6 molecules of mRNA; phospholipid cholesterol; a PEG lipid that keeps the lipid nanoparticles from aggregating; and, an ionizable lipid, that at low pH, interacts with the mRNA. Two peer-revied articles are currently available on these techniques (Sabis et al, Mol Ther, 2018; Hassett et al, Mol Ther Nuc Acids, 2019). Dr. Moore further described that mRNAs can also be nakedly injected into the heart (VEGFA) and tumours.
Finally, Dr. Moore said that in order to make a good mRNA-drug, there needs to be translation initiation fidelity, high translation efficiency, a functional mRNA half-life, and hit the correct cell type using off-logic gates (e.g., microRNAs target sites in the 3’prime UTR causing the mRNA to be degraded in case it hits an undesired cell type). All of these factors are currently being researched and developed at Moderna.
The first question in the live Q&A related to the role of RNA structure on protein translation. Dr. Moore remitted to a Moderna publication (Mauger et al, PNAS 2019), in which by fitting detailed kinetic expression data to mathematical models, they showed that secondary structure can increase mRNA half-life independent of codon usage. She further explained that higher secondary structure seems to avoid ribosomal collision and RNA degradation. Dr. Moore was then asked about computational methods used, to which she replied that they use these models to understand to the nanosecond how LNPs form and stabilize; and also, machine-learning to comprehend how different composites and structures change LNPs.
There were many questions from the audience related to the immune responses against mRNA therapeutics. Dr. Moore said that there are no adjuvants in the vaccine formulation, although the LNPs might be immunostimulatory. Responding to a following question regarding RNA stability, Dr. Moore ensured that the COVID-19 vaccine will be stable for 18 months at -200C, and then stable in the refrigerator for up to a week. In relation to the CMV vaccine, this one is presented in a lyophilized state, stable for 18 months (-200C).
The audience was curious about how naked-RNA delivery to the heart functions. Dr. Moore said that when injected in the skeletal muscle, LNPs tend to go to draining lymph nodes; although, this is not an issue in the heart muscle. The other reason for naked-RNA delivery to the heart, is that VEGFA-mRNA needs to have a very precise-localized delivery, so that no blood vessels can grow where they might create problems.
The next question related to endosomal escape and its necessity for a functional mRNA-drug. Dr. Moore said that in comparison to the competitor LNPs in the field, Moderna’s LNPs can deliver 30% mRNA into cells vs. 1-2%. They still can’t really understand the mechanism, but Moderna LNPs seem to be more efficient.
The last question regarded purity control strategy, to which Dr. Moore said that Moderna uses quality-by-design principles and multi-layer design strategy to deliver very pure mRNAs.
"To make a good mRNA-drug, there needs to be translation initiation fidelity, high translation efficiency, a functional mRNA half-life, and hit the correct cell type using off-logic gates"