September // October 2024 Progress Update
In chimerico reprogramming
This October marked the second anniversary of the first experiment ever performed at NewLimit. Two years ago, the three of us in the lab staged an impromptu photoshoot to document the first restriction digest at our headquarters.
After a minute-long celebration, we put our heads back down. It makes me proud that as we pass this annular boundary, we have more firsts to celebrate on a grander scale.
A few highlights from the first months of autumn:
+2779 TF sets tested in primary T cells
+51 TF sets that reverse the phenotypic age of T cells
+422 TF sets tested in T cell functional assays
0 → 70 TF sets tested in human hepatocytes
5 → 7 functional assays for hepatocyte age
0 → 80% efficiency for in-house LNP delivery to hepatocytes
Functional screens in primary T cells
Our Immunology program has to date discovered rejuvenating TF sets through large scale phenotypic screens, then tested these TF sets in functional assays downstream. We continued to expand our corpus over the past two months and reached an all-time-high number of TF sets tested for the quarter (2779).
We also debuted our first functional pooled screening method for the Immunology program. This approach combines a mechanism to separate functional and dysfunctional cell populations developed by our Immunology team and a low-cost, high-throughput cell profiling chemistry developed by our Read team. We defined these functional & dysfunctional populations using one of our age-discriminating T cell functional assays.
In each experiment, we deliver a pool of TFs to old T cells and each cell acquires a random subset of TFs, just like in our phenotypic screens. We then transiently activate TFs to perform partial reprogramming and segregate the cells based on their functionality. Finally, we measure which TF sets are more abundant in the functional population relative to the dysfunctional population. If a TF set appears more often in the functional cells, it suggests that reprogramming with this set improved cell function.
We found that this new assay was able to test >400 TF sets in about the same time that it previously took us to test 20, for a >20X improvement in throughput. Results from this new method were highly correlated with our previous low-throughput experiments, giving us confidence that not only do these results reproduce, but also that our new assay is accurate.
In vivo reprogramming screens in human hepatocytes
Our Metabolism program is inventing reprogramming medicines to restore youthful function in aged hepatocytes. We hope these medicines will eventually treat common diseases of aging that affect us all. The number of possible combinations of transcription factors (TFs) that might compose our medicines is massive. Our Write (Functional Genomics) & Metabolism teams have developed a way to onboard human hepatocytes to our Discovery Engine to navigate this hypothesis space at scale.
Traditionally, therapeutics companies might screen for ways to improve hepatocyte function using cancer cells or primary cells grown on plastic dishes. There are a few problems with this approach. Cancer cells are quite different from normal human biology, and primary human hepatocytes really don’t grow well in a dish, confounding results. Experiments in these settings don’t have a great track record of translating into humans.
We believe that the results of our Engine are only as reliable as the biology of our model systems. The best model of human hepatocyte biology to our knowledge is the chimeric humanized liver system developed by our SAB member Markus Grompe. In this system, human hepatocytes are engrafted into the liver of a genetically-engineered mouse and begin to regenerate, eventually resulting in a largely human tissue within a living animal.
Throughout the summer, our team leveraged the core technology of our Discovery Engine to build the world’s first pooled system in humanized livers to our knowledge. Our technology allows us to grow humanized livers where each human hepatocyte receives a transient pulse of TF mRNA from a random set of TFs within a pool. Across this “mosaic” liver, hundreds to thousands of unique TF sets are represented.
In a single liver, we can test whether each of these TF sets make old hepatocytes look young based on gene expression and act young based on their regenerative performance. We know that old hepatocytes are much less regenerative than young hepatocytes, so this functional result is a useful proxy for the restoration of youthful function.
This month, we read-out the first results from our reprogrammed chimeras. Already in the first 70 TF sets we’ve tested, we’re seeing promising signals. We have several more of these experiments in flight now, and hope to increase the number of sets we’ve tested by several fold before the year is out.
Pre-clinical models of hepatocyte age
Our Discovery Engine screens provide us the first signals to prioritize TF sets that might rejuvenate aged cells. Ultimately, we will evaluate these TF sets in a series of scalable functional assays and traditional pre-clinical models.
This September, we measured the impact of hepatocyte age across two preclinical animal models of age-related liver disease. Each of these models mimics the liver damage that can occur from modern diets. We found in both cases that aged hepatocytes were significantly more vulnerable to this damage than young cells, suggesting that rejuvenating hepatocytes may benefit patients with similar diseases.
In total, we’ve now found that aged hepatocytes perform worse than young hepatocytes in 7 distinct functional assays and pre-clinical models. With each of these data points, we’ve grown more confident that rejuvenating hepatocytes would provide meaningful benefit to aged patients.
In vivo delivery of reprogramming factors with therapeutic chemistry
Discovering TF sets that restore youthful function is only the first step in inventing a reprogramming medicine. Once we’ve discovered a payload of TFs, we next need to formulate it into a drug-like molecule. NewLimit is developing our initial medicines using LNP-mRNA technology, similar to the COVID-19 vaccines.
This October, we took the first steps to start preparing our own LNP-mRNA formulations for in vivo testing. These first molecules are only intended for research at the moment, but bringing this capability in-house has allowed us to quickly progress initial hits from our Discovery Engine to in vivo functional testing. We’re proud of our team members for quickly matching best-in-class performance for hepatocyte delivery with our internal tools.
An idea factory
History's great laboratories were designed with intention. Be it the long hallways of Bell Labs, the small cramped offices of the Laboratory of Molecular Biology, or the desks interspersed amongst the prototyping floor in the Skunk Works, form begets function within scientific institutions, as in biology.
NewLimit’s headquarters has always been amply provisioned for our work, but from the first days in the lab, we’ve had designs on modifying it to match our culture. In October, we finally completed a remodel and moved our team into refreshed space.
We stole ideas for the layout liberally.
Our new main office is elongated, mimicking Bell Lab’s classic design. We replaced every wall that wasn’t load bearing with glass, ensuring that we feel connected to our colleagues even when we’re doing deep work. Following Kelly Johnson’s guidance, we also replaced the wall dividing our offices from our labs with a floor to ceiling window, ensuring that the ground-truth of experimental work on the “shop floor” is always close at hand. We couldn’t be happier with how it turned out.
