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Dave Madge, Vice President of Discovery Services at WuXi AppTec, presents on ‘Creating a Comprehensive Therapeutic Oligonucleotide Discovery Platform’.
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Dr. Madge leads business development in Europe and Israel and oversees strategic initiatives across multiple therapeutic areas and modalities. Before joining WuXi in 2014, he was VP of Research for the ion channel drug discovery company, Xention, in Cambridge, UK, developing new molecules for cardiovascular and respiratory disorders.
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Thank you for the introduction and for being here this morning. I'll spend 20 minutes discussing the discovery platform we've been building over the last four or five years for oligonucleotide therapeutics at WuXi. I'll start by explaining our approach to drug discovery, platform building, and enabling organizations like yours.
1:28
We focus on understanding disease, targets, and pathways, and matching these to modality selections and discovery platforms. We discussed making appropriate choices within the oligonucleotide discovery space for different types of oligos. The landscape is broader, and we work across all modalities shown in the slide.
2:11
Our sister company, WuXi Biologics, supports antibody and ADC discovery. We also have a division for cell and gene therapy, but I'm here representing our small molecule business, which includes small molecule peptide macrocycles and oligonucleotide therapeutics.
2:31
Our philosophy is to understand the science required to move from target selection to patient treatment. We build platforms that collaborators and clients can plug into at any stage of the pathway, creating an end-to-end platform for drug discovery across many modalities and therapeutic areas.
3:20
Today, I'll focus on oligonucleotide therapeutics. We have opportunities to intervene at different stages of mRNA and protein production with various types of small and large oligonucleotide therapeutics and conjugation methods. Although we still have challenges in delivering these selectively to specific cells, the landscape is expanding with more opportunities.
4:19
The landscape includes RNA-targeting small molecules, with significant investment and investigation in different therapeutics for oligos. There is a lot of work in this area, with many new and established companies developing numerous molecules.
5:08
We develop a workflow screening cascade to discover targets, design oligos, characterize molecules, and build a short iterative loop for optimization. I'll exemplify this for small interfering RNA therapeutics, but many building blocks are common to different oligonucleotide and small molecule therapeutic discoveries.
6:04
The four main building blocks in designing and optimizing molecules are sequence selection, topology, modifications, and targeting. We start with sequence selection, predicting which sequences will provide on-target activity and minimize off-target activity. We then translate this into chemistry, selecting modification patterns to achieve stability, selective delivery, and minimize off-target liability.
9:26
We consider conjugation methods and targets such as ASGPR, lipid receptors, peptide receptors, and aptamers. We need to make these molecules on different scales at various stages of discovery, from small libraries to multi-gram scale synthesis.
10:02
We need to be able to make large libraries very quickly. Typically, we generate 100 to 200 double-strand sequences, which means up to 400 single strands before annealing. This requires multi-channel synthesizers, up to 192 channels, allowing us to produce these small libraries in three to four weeks after purification and annealing.
10:30
Initially, this happens on a very small scale to enable high-throughput in vitro assays. We then move to larger scale synthesis, up to small milligram quantities, using more dedicated single-channel or lower-channel number equipment. Ultimately, we scale up to multi-gram synthesis. My colleague Pete discussed taking these molecules into much larger scales, including GMP and commercial manufacture. Today, I'll focus on discovery.
11:14
We discussed different flavours of GalNAc conjugation for hepatic-specific delivery via ASGPR. This well-established area of chemistry allows us to deliver double-stranded and single-stranded oligonucleotides selectively.
11:34
The chemistry is complex, involving many steps to build the conjugate before joining it to a linking molecule and then to the oligo itself. Making these molecules on scale is not trivial, but the chemistry is well worked out and can be carried out on kilo and GMP scales. We've made thousands of molecules with this type of conjugation.
12:04
Similarly, lipid conjugation for oligo delivery offers various flavours, some providing good permeability across the blood-brain barrier, others targeting specific cell types or aiding distribution, bioavailability, and stability. Our chemistry teams are well-versed in conjugating these by small linking groups to different oligos.
12:49
Increasingly, we work with oligonucleotides conjugated to peptides. There are neat ways of identifying peptides selected for cell surface receptors, which can target oligos to specific cell types where those receptors are upregulated in disease.
13:13
The ability to conjugate peptides to oligos with robust maleimide or click chemistry is well established in our labs. This is exemplified with IDD cyclic peptides and analogues of GLP-1R targeting peptides for delivering oligo therapeutics to specific cell types, such as pancreatic cells in the case of GLP-1.
13:50
Turning to biology, we want to characterize these molecules quickly and accurately in a way relevant to the disease and mechanism we're targeting. We have many stable human cell lines available and can create new cell lines or genetically engineer them for specific mutations. For siRNA therapeutics, we look at mRNA downregulation and subsequent protein production reductions.
14:31
Genetic tools like qPCR, branch DNA, and Sanger sequencing help us understand the degree of mRNA knockdown in different cell types. We can do this with transfection or free uptake, depending on the conjugate and discovery program stage. ELISA and bead-based methods like CBA are well-established for protein level analysis, giving us a quick and accurate way to assess mRNA and protein knockdown longevity.
15:28
We also use reporter assays, such as psiCHECK assays in human hepatocytes, to evaluate functional effects. Our platform enables us to assess the functional consequences of protein downregulation in various cell types, providing a comprehensive in vitro characterization package.
16:13
For in vitro studies, we typically create small arrays of a few hundred oligos, starting with RT-qPCR assays at a couple of concentrations and moving to dose-response curves. We often use a second method, like psiCHECK, to cross-reference data sets. Having a reference point, around 700-800 in the top right data, helps us analyse initial screens of siRNA molecules with free uptake. We then move to dose-response curves for molecules showing more than 50% inhibition.
17:23
For more functional evaluation, we use various tools in our labs to build integrated screening cascades involving functional assays in different cell types for on-target and off-target activity.
17:54
In vivo studies are crucial for translational research. Many molecules are designed to target human genes, which often don't cross over to other species. We use tools like humanized mice, hydrodynamic injection, viral vectors, and transgenic models to represent human genes in animal models. Humanized mice, such as the FRG surgical mice model, are the gold standard.
18:44
Hydrodynamic injection is a quick way to establish short-term human gene expression, while viral vectors offer a longer window for in vivo studies. Transgenic models take time and effort but provide a good way to study long-term gene knockdown in vivo. We typically use a blend of these methods, starting with HDI for quick in vivo signals and moving to transgenics and FRG humanized livers for long-term targets.
19:40
We also have access to an established non-human primate colony for in vivo knockdown studies, providing high-quality data on protein levels in plasma and mRNA knockdown in the liver.
20:29
To move these molecules safely into human studies, we assess cytotoxicity, cell viability, apoptosis, and immunogenicity. High-throughput immunogenicity assays help us identify potential issues. Off-target profiling by RNA-seq ensures selectivity, generating volcano plots for analysis.
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For bioanalysis, we focus on high-resolution mass spectrometry and qPCR. There are excellent talks on analytical techniques for oligo therapeutics later this morning.
21:59
A case study on lowering LDL cholesterol using PCSK9 as a target demonstrates our workflow's effectiveness. We selected 200 double-strand sequences, identified active molecules, and conducted in vitro and in vivo studies, resulting in potent, selective, and in vivo active molecules. This workflow produces high-quality data quickly, enabling us to identify molecules with similar in vivo efficacy to the current clinical gold standard.
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Our end-to-end platform supports organizations of all sizes, allowing them to plug into our workflow and drive their science forward. Thank you for your attention.
