Despite leaps in therapeutic advancements over the past few decades, there are many rare diseases without good treatment options for patients. One reason is that most rare diseases are caused by an array of mutations, rather than one predominant mutation (sometimes described as a “founder effect”). In many rare diseases, those different mutations can cause the same disease through slightly different mechanisms of action, making it very challenging to develop one molecule to address a significant portion of the mutations that cause the disease. The Octant Navigator platform is pioneering new multi-target therapies to address this challenge using an approach to drug discovery we call Broad Target Scanning (BTS). In this blog post we’ll walk you through BTS, its application to one of our drug programs, and the synthetic biology of designing reporters for assays that support BTS.
Broad Target Scanning is the process by which we screen molecules for their ability to exert drug-like effects on tens to hundreds of targets simultaneously. In the case of rare diseases, rather than carrying out one experiment for each variant of a causal gene in series, we examine interactions between compounds and variants in parallel. We engineer cell lines to each contain a variant of the target paired with a unique DNA barcode. Thanks to our high-throughput synthetic biology capabilities we can make hundreds of these unique cell lines within a few weeks. Once the cell lines are built we can pool them for experimentation because each cell type is tracked by a unique barcode that identifies biological activity from that cell. The more a particular downstream activity of the gene in that cell is activated, the more barcode is produced by the cell. We iteratively synthesize and test libraries of chemicals and screen them against these cell-systems in order to identify and optimize promising compounds to treat the dysfunction.
One application of BTS to our drug programs is in our Rho-associated autosomal dominant Retinitis Pigmentosa (Rho-adRP) program where we are striving to develop a best-in-class orally bioavailable therapy. Rho-adRP is a genetic disease characterized by degeneration of the rod photoreceptors of the retina. It initially presents as night blindness, spreading to cone photoreceptors, leading to loss of peripheral and eventually central color vision. The treatment options for patients with Rho-adRP are bleak, with no current approved therapies.
We could offer a therapy to thousands of patients with this disease if we could engineer drugs for Rho-adRP that act on a broad set of disease causing mutations. In healthy individuals, rhodopsin is trafficked to the cell surface, but for Rho-adRP patients misfolded rhodopsin protein is mistrafficked by the cell, causing toxic build-ups that eventually lead to cell death. There is evidence that assisting these proteins to fold correctly with a small molecule corrector or chaperone could clear these toxic protein traffic-jams and relieve the burden of the disease.
We set out to build a multiplexed BTS assay that could evaluate our molecules against many different pathogenic variants of the rhodopsin protein in parallel (for more scientific detail on this assay, make sure to read the Scientific Deep Dive down below). The results from the multiplexed assay were encouraging. Below is a small sample of data from one of our screens that demonstrates molecules attaining potency at different levels (potency increases from blue -> yellow) against various clinically relevant rhodopsin variants.
And we’re not stopping with adRP. We’ve built a repeatable strategy for identifying pan-mutant chaperone therapeutics across several diseases caused by the misfolding and mistrafficking of proteins. Because these diseases are driven by similar cellular mechanisms, repurposing our synthetic biology toolkits across different causal genes is a rapid and effective way to search for high-value pharmacology across different indications.
If you’ve made it this far you might be as big of a synbio nerd as we are! In this section we’ll walk you through how we built a generalizable multiplexed reporter that we used to evaluate potential chaperones for multiple pathogenic rhodopsin variants.
Initially, for Rho-adRP we performed primary screening on a cell line harboring the P23H mutant of rhodopsin – the most common variant associated with Rho-adRP estimated to comprise ~15% of pathogenic alleles.1 This assay utilized a split luciferase reporter system composed of a small bit (HiBiT) and a large bit (LgBiT) that could measure the degree to which the P23H rhodopsin variant reached the cell surface. In this assay a complementation event between the extracellular large bit, which is added in trans, and the small bit, which is fused to the N-terminus of P23H rhodopsin, generates light. Luminescence of wells can be quantified on a plate reader. Higher photon counts signify more instances of the complementation event, which acts as a proxy for the amount of mutant rhodopsin trafficked to the surface. This assay can thus be used to evaluate the efficacy of different therapeutic chaperones.
Once we achieved satisfactory potency of our compounds against the most common variant, we built a multiplexed assay to report on the activity of several variants. Rather than measuring light, we built an assay where barcode transcription is the output, since the number of these distinguishable probes is nearly infinite. This enabled us to measure the effects of our chaperones on multiple different mutations in a single well. Historically, we’ve used multiplexed assays where a signaling event leads to the expression of a barcode, but here things are different since we’re looking at trafficking. So the big question for us was, “How do we pair the trafficking of rhodopsin variants to the cell surface with the expression of a unique barcode?”
To accomplish this, we engineered a system with a membrane-anchored protease that controls barcode expression. In this system, rhodopsin is engineered to contain a cleavage site that can be recognized by the membrane-anchored proteases. On the other end of this cleavage site is a transactivator fused to a DNA binding domain. This DBD-TA can selectively bind the barcode promoter and initiate transcription. When rhodopsin variants get trafficked to the cell surface they present the cleavage site within proximity of the protease to cut it, releasing the DBD-TA to express the barcode and allow us to measure trafficking of many variants in multiplex.
This genetic circuit was the end product of many different tests performed on hundreds of unique stable cell lines. To ensure we were achieving the best reporter performance we optimized many genetic elements of our system including the membrane anchor, protease, linker between the anchor and protease, DBD, promoter sequence and strength, and seven other genetic components. Many of these components made or broke the assay, so being able to test them all in combination with each other allowed us to leave no stone unturned in developing a high-fidelity assay. For example, the DBD choice was one of many parameters that strongly influenced the assay’s sensitivity, which can be seen in the image below. Without an ability to test a wide matrix of components, we could have easily overlooked synergy between genetic elements that led to dramatically improved assay performance.
For validation, we benchmarked the assay by comparing the results from the multiplexed assay to the results from the single channel split luciferase assay described above. It was gratifying to confirm that our multiplexed assay was concordant with the split luciferase assay!
BTS enables us to examine how molecules rescue the function of many pathogenic rhodopsin variants in search of pan-mutant correctors for Rho-adRP. A more rational approach to drugging the largest possible patient population can be a significant game-changer for thousands of patients. BTS also enables us to run more informed clinical trials informed by data on which genotypes are likely amenable to our therapy. While we’ve focused in this blog post on solving Rho-adRP, BTS offers a novel way to discover small-molecule therapies for numerous other rare diseases with diverse genetic etiologies. We’re harnessing this groundbreaking technology with the hopes of improving the quality of life for thousands of patients.