Unexpected PLOD1 twist

Collagen is a slippery eel. After trying and failing to visualize collagen inside PLOD1-kEDS fibroblasts, we pivoted to measuring collagen secreted by cells as a readout for a drug repurposing screen.

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Perlara rebooted as a remote first, fully distributed biotech company in Q4 2021. The first pioneer family to engage us as scientific consultants — Cure Guides — asked us to develop a personalized cure roadmap for their child living with an ultra-rare form of Ehlers-Danlos Syndrome (EDS) caused by deficiency of the enzyme PLOD1, which is required for proper collagen production by the body.

In that roadmap, we proposed a cell-based drug repurposing project as our main objective. We thought the project would take at most two years to complete. We enter the third year of this cure odyssey guided by the same north star, and lugging a suitcase full of accumulated scientific and operational learnings. Most importantly, we have a line of sight on what appears to be a robust assay that would finally enable us to consummate a high-throughput drug repurposing screen.

There’s still no guarantee we will pull it off, but we have confidence in our plan. Perlara Cure Guides L Handy and Dr Shiri Zakin have been working with Dr Yoshi Ishikawa at UCSF over the past year to develop a cell-based assay for drug repurposing for PLOD1-kEDS. Last summer, L completed proof-of-concept of experiments that green lit advancing to where we are today.

Rewinding the clock back to 2022, the first major roadblock was not being able to visualize a statistically discernible defect in collagen fiber structure inside patient fibroblasts cultured in the lab. Those cell imaging studies are summarized here. Last year, we pivoted to a biochemical strategy: measuring levels of extracellular collagen secreted by PLOD1-deficient cells. PLOD1 adds hydroxyl groups to lysines in the collagen protein, encouraging cross-linking between collagen strands that can be detected by antibodies in a conventional ELISA assay.

First, however, we had to establish the specificity and fidelity of our PLOD1-kEDS research toolkit: PLOD1-deficient mouse and human cell lines; antibodies specific to different types of collagen. The launch phase of our collaboration with Yoshi is described here for the collagen aficionados in the audience.

Preliminary results were encouraging but we wanted to run additional controls to make sure we were interpreting the results correctly. Yoshi tested two different antibodies specific for lysyl hydroxylase 1 (LH1), the protein encoded by the PLOD1 gene. No LH1 protein was detected by immunoblot in two different PLOD1-kEDS patient fibroblast lines obtained from the nonprofit biobank Coriell: a line derived from an affected infant and an older child’s line.

C = control; P = patient. G159 is the location a pathogenic nonsense mutation.

Next, Yoshi biochemically surveyed the maturation of collagen fibers by immunoblotting with a panel of antibodies. Type 1 and Type 5 collagen are significantly reduced in the two PLOD1-kEDS patient lines compared to the two control lines. On the other hand, Type III collagen appears to be spared or modestly decreased in the two PLOD1-kEDS patient lines.

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The two LH1-specific antibodies tested both target an epitope close to the C-terminus of the protein. We would expect to see the same result using an LH1-specific antibody that binds to a stretch of amino acids near the N-terminus (opposite end) of the protein. Indeed, the N-terminal-specific anti-LH1 antibody failed to detect any LH1 protein.

This cure odysseys has taken unexpected twists and turns, much like an errant collagen fiber. The story made a sharp and sudden 90-degree turn when Yoshi measured LH1 protein levels by immunoblot in the PLOD1-kEDS family trio. LH1 levels are reduced in mother (“M”), father (“F”) and affected child (“P”), but notice how the paternal and maternal banding pattern is different.

The original genetic testing report concluded that the affected child is homozygous for a nonsense variant in the fifth exon of the PLOD1 gene. But Mom and Dad do not carry the same PLOD1 genotype according to LH1 biochemistry!

Let this serve as a lesson in edge cases, which are more common than we realize. Clinical geneticists often will insist on having a DNA sequencing test result and a corroborating protein activity test result that mutually agree. A possible genetic explanation for the disagreement we observe is that the father actually carries a large deletion that is invisible to old-school PCR-based genetic tests.

The large deletion hypothesis was suggested in a follow-up genetic report generated by the Collagen Diagnostic Laboratory (CDL). The report acknowledges that there could be a multi-exon deletion that would not be detected by standard gene sequencing.

We’re sorting out the exact genotype of the PLOD1 locus to confirm the presence of a paternal multi-exon deletion variant.

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After ruling out the feasibility of a LH1 enzymatic activity assay, we narrowed on the final option: a biochemical assay to measure extracellular collagen secreted by PLOD1-kEDS patient fibroblast lines grown in a multi-well-plate format amenable to high-throughput screening. This schematic summarizes our proposed workflow.

Before using the precious patient samples, we optimized the hydroxylysine assay using purified collagen from mouse fibroblasts. Right off the bat, we ran into assay development challenges. We quickly learned that the first hydroxylysine antibody we chose was incompatible with an ELISA use case. We found a new antibody that binds to human, mouse and bovine Type I collagen. Problem solved. L ran the first successful proof-of-concept experiment right before the holidays.

The binding of non-hydroxylated collagen purified from the LH1 knockout mouse cell is reduced by 50% compared to the control collagen purified from a wildtype mouse cell line. We finally found an assay that we could deploy in a drug repurposing screen. We also validated a functional cell-based assay for differentiation between hydroxylated and non-hydroxylated collagen that anyone of the EDS space can run with beyond our immediate focus on drug repurposing.

Yoshi, L and Shiri have come up with a scope of work that would take us through the rest of assay development. To minimize unnecessary depletion of patient fibroblasts, Yoshi is creating a LH1 knockout human cell line for the final stages of assay development and possibly for use in a drug repurposing screen itself if we elect to save patient fibroblasts exclusively for hit validation studies. More to share soon!

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