A look back at RNA Therapeutics – day one

RNA molecule

On Wednesday 8th February, leaders and experts from across life sciences made their way to the picturesque Copthorne Tara Hotel in Kensington, London, to attend the 14th Annual RNA Therapeutics Conference.

Only a stone’s throw away from both of the city’s famed tributes to innovation and scientific discovery (the National History Museum and Science Museum), the location provided a fitting backdrop for speakers to investigate the next generation of genetic medicine through RNA-based therapies.

With guests travelling in from around the world to attend this return to in-person events, the thirst for knowledge and sharing of ideas was palpable. And so, with coffee sipped and laptops primed, the stage was set.

Pre-lunch talks

After a brief welcome introduction, conference chair and BioNTech VP Formulation & Drug Delivery, Heinrich Haas, welcomed the morning’s first presenter Shalini Andersson, vice president oligonucleotide discovery for AstraZeneca, to discuss the opportunities and challenges of developing oligonucleotides into therapies.

Recent advances in genomics have opened up myriad new drug targets for oligonucleotides that selectively suppress target genes, she explained, however, progress in translating the potential of these agents has been somewhat limited by barriers in achieving efficient targeted delivery.

The problem is, these highly delicate short-lived strands of genetic material are often more hydrophilic and larger in size than small molecule agents, which are readily taken up by cells. As such, cellular uptake remains a prominent barrier for oligonucleotides research. To successfully deliver the mRNA to the intended target for protein expression, it needs a protective shell of sorts to shield it from destructive enzymes. Currently, Andersson explained, the gold standard for efficient delivery of mRNA is lipid nano particles (LNP). However, she notes, repeated dosing of mRNA can result in an immunogenic response to LNP.

Optimising LNP formulations is a key focus for researchers. With the right LNP, scientists can deliver better expression in as low a dose as possible. To demonstrate how this can work, Andersson pointed to results from a study into the targeted delivery of a promising liver-targeted N-acetylgalactosamine (GalNAc) conjugated antisense oligonucleotides (ASOs), which allowed researchers to reduce the dosage tenfold in healthy volunteers. This is a particularly important element, she explained, as “patients don’t want large dosage volumes if they can avoid it”.

Looking to the opportunities for oligonucleotides, Andersson pointed to the significant impact that preventing mRNA from being translated into a detrimental protein product could have for the treatment of genetic drivers of disease. “Our aim is not just to manage disease, but to do something about it,” she concluded.

Her presentation was, as Haas described, a “perfect introduction to the conference”. And, with the challenges and potential of mRNA therapeutics now firmly at the forefront of attendee’s minds, Haas took to the lectern to begin his talk on tailoring delivery systems for next generation mRNA therapeutics.

While BioNTech may have gained mainstream public attention for its work in developing vaccines for COVID-19, Haas was clear to note that it is still a cancer and immunotherapy focused company. With that said, he dove into the recent history of liposome and nanoparticle development, beginning with Alec Bangham’s discovery of liposomes back in 1964. Today, he noted, at least eight liposome formulations have been approved by the FDA and EMA for cancer therapy, a number that pales in comparison to the number of research papers on the subject – “So many papers, so few drugs on the market,” quipped Haas.

Echoing Andersson’s presentation, Haas explained that “LNPs are now the password for RNA delivery […] by choosing the right lipid composition and manufacturing, I can tailor the delivery system to the need.”

Formation, he said, is particularly important. To illustrate this, he spotlighted three key assembly protocols: Lipoplex nanoparticles (LPX), formed from liposomes and RNA in aqueous buffers, LNPs formed from lipids in ethanol and RNA in aqueous buffer, and polyplex nanoparticles (PLX), formed from polymers and RNA in aqueous buffers. Choosing the right lipid composition allows researchers to select the properties needed for the intended application.

For Haas, the greatest challenges for the future of RNA will be delivery. To maximise the potential of tailored formulations, researchers will need to develop an in-depth understanding of the required particle characteristics, including composition, structure, charge, and release, as well as advanced characterisation methods to aid rational formulation development.

Chromatography in mRNA production: from analytics to purification

To begin his presentation, Rok Sekirnik, head of process development, mRNA pDNA at Sartorius BIA Separations took a moment to acknowledge the number of different expertise and approaches represented in the room.

Turning his attention back to the task at hand, Sekirnik provided a picturesque description of the company’s base of operations in Vipava Valley, Slovenia. The area, he explained, is a well-known wine region. Aside from the obvious appeal of this surrounding landscape, Sekirnik highlighted a key link between the operations taking place within each industry. Expert winemakers have mastered elements that pharma researchers have long struggled with, such as chemical stability and acidity.

When it comes to mRNA, optimising yield is highly important. In vitro transcription (IVT) – a method by which researchers can obtain highly pure and uniform mRNA oligonucleotides of lengths ranging from about 15 to several thousand nucleotides – is one of the most expensive steps in the mRNA production process. As such, Sekirnik noted, if you want to keep costs at a minimum, IVT reactions are a prime place to start.

It is here, he explained, where chromatograms can play a valuable role. He pointed to Sartorius’s CIMac PrimaS to demonstrate how real time monitoring of IVT reaction improves process control. By monitoring mRNA production, kinetics researchers can identify maximum productivity and prevent degradation. Furthermore, by combining a fed-batch concept with co-transcriptional capping would allow scientists to pair manual feeding with automated feeding, thereby achieving a steady state of nucleotide consumption without overfeeding the mixture. With this, he concluded, researchers could save between 25% to 50% of RNA.

Targeting neurological disease

Following on from Sekirnik, Oliver Ernst, managing director for NEUWAY Pharma GmbH, made his way onstage to present ‘mRNA delivery to the CNS: targeting neurological disease’.

He opened by describing the challenges associated with treating CNS diseases. The human body, he explained, has established protective barriers to act as a structural and functional roadblock to stop microorganisms, such as bacteria, fungi, viruses, or parasites, from accessing the CNS. However, this is a challenge for researchers working to treat CNS diseases, as these barriers also prevent many therapeutics from reaching their intended targets.

Delivery, he emphasised, is the real limitation. To overcome the challenge of navigating the brain blood barrier, NEUWAY is using its Engineered Protein Capsules (EnPC) to develop an entirely novel class of biotherapeutics. Highly monodisperse EnPCs protect therapeutic load and eliminate cellular uptake competition. Moreover, he explained, mRNA encapsulated in EnPCs can remain stable for at least one month at 4oC and three months when stored at -80 oC. As such, delivery of treatments can be flexible.

“Our goal is to make this treatment available to all patients – not just patients that can afford it,” he said.

Sticking with the therapeutic potential of mRNA, Daniel Tondera, head of biology at Pantherna Therapeutics, was next to the podium to deliver his presentation on ‘The therapeutic potential of mRNA in the restoration of endothelial cell function’. With a focus on acute respiratory distress syndrome (ARDS), Tondera explained that the goal of mRNA treatments in this area is to prevent the development of lung oedema during the acute phase of the disease, thereby reducing the need for the patient to be placed on a ventilator.

To address this, Tondera detailed Pantherna’s work to develop a spatial Tie2 receptor agonist for restoring capillary endothelial function in the lung. With PAN004, he explained, the company has developed an mRNA-LNP delivering an mRNA encoding a Tie2 activator selectively at the site where lung oedema occurs.

Addressing challenges in RNA delivery

The final presentation for the morning came from Senne Dillen, drug product & substance senior scientist at eTheRNA immunotherapies NV.

After a round of thanks for both presenters and attendees for their presence, Dillen opened his talk with a twist on a famed Jane Austen quote:

“A company in possession of a good idea, must be in search of a partner,” he joked.

While it may have been light in tone, his statement is not far from the truth. An idea, he explained, is a great starting point, but an idea alone will not get you very far. “It doesn’t make sense to have a good RNA therapeutic if you are not able to deliver it,” he said.

Hurdles for RNA therapies are numerous and diverse, he explained, ranging from broad and undirected LNP biodistribution, poor understanding of what makes an effective RNA construct, to critical cold chain storage and distribution requirements. COVID-19 vaccine efforts are a key example of this, as storing the vaccine emerged as a particular challenge.

It is possible to overcome these hurdles, he explained, pointing to etheRNA’s work to develop RNA with low levels of process and product-related impurities, using proprietary codon optimisation and UTRs to create RNA constructs for maximum efficacy. Moreover, he concluded that, with the company’s robust lyphilisation processes, RNA LNPs could be stored at standard refrigerator temperatures.

The role of clinical pharmacology in RNA modalities

After a brief respite for lunch, where attendees congregated to discuss the mornings events over an array of coffee mugs and dinner plates, we were ushered back into the main conference room to begin the afternoon’s presentations.

First up to the stage was Venkatesh Pilla Reddy, director of clinical pharmacology and pharmacometrics for AstraZeneca, to talk us through the role of clinical pharmacology in the development of novel RNA modalities.

But what exactly is clinical and quantitative pharmacology? For Reddy, it contributes a large percentage of the information found on drug labels. Quantitative data provides stakeholders with vital information to help them understand drugs, diseases, and their mechanistic relationships.

There are a number of factors that contribute to how a patient responds to a therapeutic. These include safety concerns, such as injection or infusion reactions as well as intrinsic and extrinsic factors that may lead to clinically relevant differences in the response to RNA therapeutics.
By modelling and assessing these factors, clinical pharmacology can help to identify the right dose, regimen, and patient for each therapeutic.

AI and machine learning in RNA therapeutics

As we neared the end of the day, attention turned to another buzzword catching the attention of life sciences at the moment: artificial intelligence.

Talk of AI and machine learning in RNA began with head of RNA Therapeutics for Abzu Aps, Lykke Pedersen’s presentation, ’Drug and target discovery with explainable AI’.

Explainable AI, as Pedersen described it, looks to address the relationships and connections to be found between the numbers. It begins, she explains, with asking the “why” questions.

“If we look towards the more traditional AI models, these are less adept at addressing why questions, such as why did you get a disease, they are better at identifying whether or not something will happen,” she explains. “We are trying to give you answers instead of numbers.”

This is where Abzu Aps works to translate data models into more digestible language. Pederson illustrates this process by showing how the company can break down RNA Tx into descriptors, or sequences.

With explainable AI, she said, researchers can identify drug targets and improve drug design.

Taking up the mantle of AI discussions, Martin Akerman, chief technology officer for Envisagenics was next up to the microphone. During his presentation, ‘AI/ML developed RNA therapeutics for neurodegenerative diseases’, he focused on the company’s SpliceCore technology for drug discovery.br /> The spliceosome, he explained, is on of the largest molecular machines in the cell. With more than 300 proteins. It is highly dynamic, however, it also has the highest rate of errors. Splicing errors cause or aggravate 370 human diseases. In fact, eight of ten top driver RNA-binding proteins in neurodegenerative diseases have been shown to either regulate alternative splicing, participate in spliceosome biogenesis, or co-precipitate with core spliceosome subcomplexes, he explained.

Looking at ALS, he noted that 10% of patients are ‘familial’ or have a known genetic cause, however, 90% of patients are ‘sporadic’, with an unknown genetic cause. This is where AI and machine learning can help to identify patterns in patients, which may uncover druggable targets.
“Given the importance of splicing in familial ALS patients, the sporadic patients can be stratified by recurrent patterns of deregulated splicing factors – which can then be explored as potential drug targets.”

When tasked with profiling the spliceosome of ALS patients, SpliceSlice was able explain 37% of sporadic ALS pt samples, and predict errors in five sub populations, noted Akerman.

Diversification is also a highly important factor when selecting a drug target, he explained: “In general, we do something called a diversified approach – more than one method of target selection – disease specificity, function probability, what is druggable.

“What you get is a diverse target – like the stock market, you don’t want to put all your eggs in one basket.”

Finally, to close out the day, the last speaker Yaniv Erlich, CEO of Eleven Therapeutics, took the mic to discuss optimising RNAI applications through novel AI technologies.

“If you want to do AI,” he began, “the dirty secret is that you need a lot of data.”

Using this statement as a springboard, Erlich dove into the body of his presentation, which he separated into two sections. The first was dedicated to discussing the benefits of DNA encoded libraries (DEL). Whereas drug discovery typically involves investigating one target at a time, DEL technology allows researchers to synthesise and screen millions of encoded compounds in a faster and most cost-effective manner. Erlich likened the process to trying to find a needle in a haystack. With conventional methods, scientists are searching the stack straw by straw; DEL allows them to scan the entire stack and zone in on the hidden target.

For the second part of his presentation, Erlich spotlighted Eleven Therapeutic’s TERA platform. To achieve strong production of mRNA, you have to modify it, he noted. There are more than 100 different types of RNA modifications, but these need to be precisely placed to maximise their impact. To address the challenges of RNA modification, the company has developed TERA, a DEC, which works by splitting ‘beads’ at random into different synthesis columns and incubating them with cells and a DNA barcode that records the modifications. Once the modified RNA oligos and DNA barcodes are release from the bead into the cell, researchers can measure their effect in each cell using a high throughput assay.

He concluded by thanking attendees for their attention and emphasised the promise of ideas and technologies shared during the day’s presentations.

And with that – day one of RNA Therapeutics came to a close. Stay tuned for our upcoming coverage of day two.