Development and Validation of a Gamma Globin RT-Ddpcr Exploratory Biomarker for Sickle Cell Phase 1 Clinical Trial Fis 002-2020

FTX-6058, a selective and potent binder of embryonic ectoderm development protein (EED), is being investigated in Sickle Cell Disease (SCD). In-vitro and pre-clinical Townes mouse studies show FTX-6058 upregulates expression of the gamma globulin gene leading to increased levels of fetal hemoglobin (HbF). In human clinical trials, as an early indicator of FTX-6058 induced gamma globulin expression, a reverse transcriptase droplet digital PCR (RT-ddPCR) assay was developed and validated by Precision for Medicine. Versus RT-qPCR, RT-ddPCR gives absolute copy number, does not require a standard curve, and is more precise and reproducible for low expressed targets 1,2. Precision for Medicine developed and validated RT-ddPCR assays for the target globin genes α, β, and γ and for the housekeeping genes TFRC and OAZ1. RNA isolated from 3 healthy and 3 SCD affected individuals was used for development and validation of the assay. The upper and lower limits of quantification were 120,000 and 10.5 copies/20μL reaction, respectively. For samples with >50 copies/20μL reaction, the CV for technical replicates was <20% for all transcripts. Furthermore, Precision for Medicine demonstrated SCD affected individuals express approximately 15-fold more γ globulin versus non affected individuals. Fulcrum utilized the globin RT-ddPCR assay validated by Precision for Medicine in the FTX-6058 phase 1 clinical trial FIS 002-2020. 1 Zhao Y, Xia Q, Yin Y, Wang Z (2016), Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri. PLoS ONE 11(7): e0159004. doi:10.1371/journal.pone.0159004 2 Taylor SC, Laperriere G, Germain H (2017), Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports 7(2409): DOI:10.1038/s41598-017-02217-x Disclosures Roth: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Yang: Fulcrum Therapeutics, Inc.: Consultancy. Tsavachidou: Fulcrum Therapeutics, Inc.: Consultancy. Davis: Fulcrum Therapeutics, Inc.: Consultancy.