PLOD1-kEDS research update
What began as a CureMap morphed into our first popup project. After multiple rounds of assay development, we're ready to finish the pivotal therapy-enabling experiments we recommended last year.
A year ago we had just completed the PLOD1-kEDS CureMap on behalf of an entrepreneurial family with an affected child. As often happens on the uncharted frontiers of science and medicine, we had to adapt to unexpected obstacles along the way.
Out of the gate we spent several months last winter vetting contract research labs that were ultimately too expensive, too inexperienced or simply uninterested in working with us. In one instance, an academic core facility with the requisite skillset ghosted us after several weeks of encouraging email correspondence.
By last spring, we concluded the CRO sweep and made the determination that Perlara needed to pop up a lab to perform a series of “killer experiments” on patient fibroblasts. Enter SFBiolabs, a turnkey startup incubator space in the heart of San Francisco, and the flagship node in Perlara’s distributed cure lab network.
By May of last year we were ambitiously planning to complete experiments by the fall on the assumption that our family’s child’s fibroblasts would be ready to ship from Coriell on the original timeline communicated to us.
After waiting many months and the estimated delivery date passing, we pivoted and ordered the PLOD1-kEDS patient fibroblasts that were already available for distribution. At least we could use the down time to iron out assay conditions, we thought.
There wasn’t much published on collagen phenotypes in PLOD1-kEDS patient fibroblasts but we had reason to believe that we could see differences in the directional orientation of collagen fibers in extracellular matrix deposited by fibroblasts growing in culture for 6+ weeks.
Wildtype mature collagen fibers are oriented in one direction. Collagen fibers produced by PLOD1 deficient fibroblasts are expected to be more disorganized, and so oriented in two or more directions.
Over the summer we worked out collagen staining conditions using the existing PLOD1-kEDS fibroblast lines. Last October we finally received our family’s patient’s fibroblasts, and by December we were finally able to collect microscopy images.
The desired result was a stunningly obvious and statistically significant visual difference between the patient line and the control line. Unfortunately, the results are inconclusive.
When you hit an impasse in research, you either stop or find a way forward around the obstacle. So we pivoted again, this time from microscopy to biochemistry.
There are two ways to measure PLOD1/LH1 lysyl hydroxylase activity in patient fibroblasts without directly visualizing or physically interrogating collagen fibers. Measure the hydroxylysine content in a cellular sample, or measure the conversion of an artificial substrate by partially purified LH1 enzyme from a cellular sample. Both assays should converge on the same answer, in theory.
We plan on performing the above biochemical assays on our family’s patient’s fibroblasts over the next 2-3 months in collaboration with a PLOD1 expert at UCSF.
Ideally, one or both biochemical assays will be miniaturized for high-throughput drug multipurposing screens. In parallel, we will test proof-of-concept exon-skipping ASOs and proof-of-concept enzyme replacement constructs in one or both biochemical assays. In the best case scenario, the resulting data package will enable the transition from project to startup.
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