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ASHG Education Event

Thursday, October 19


12:30 to 1:45 PM
Lunch will be served
Hilton Orlando Hotel
Lake Eola, Lobby Level


ASHG Education Talks
:

Rapid, Non‑Invasive Detection of Lung Transplant Rejection from Donor‑Derived Cell‑Free DNA

Droplet Digital™ PCR to Evaluate Systematic Gene Tagging with CRISPR/Cas9 to Illuminate Stem Cell Organization and Dynamics

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2017-10-19 15:30:00 2017-10-19 04:45:00 America/New_York ASHG Bio‑Rad Laboratories Exhibitor Education Event Hilton Orlando Hotel, Lake Eola, Lobby Level
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Rapid, Non‑Invasive Detection of Lung Transplant Rejection from Donor‑Derived Cell-Free DNA

Andrew Young
Washington University in St. Louis


Fiberoptic bronchoscopy and transbronchial lung biopsy are currently the gold standard for detection of acute rejection following human lung transplantation. However, these surveillance procedures are expensive and invasive. Here, we present a novel method for the early detection of lung allograft rejection by quantifying donor‑derived circulating cell-free DNA (cfDNA) relative to recipient‑derived circulating cfDNA using Droplet Digital™ PCR (ddPCR™). First, we developed a panel of probes, each targeting a unique sequence of the human leukocyte antigen (HLA) allele. After transplantation, donor/recipient specific probes were chosen based on the mismatched HLA loci. This enabled us to accurately measure the trace amount of donor‑derived cfDNA present within the recipient‑derived cfDNA background. To validate our methodology, we serially diluted known quantities of cfDNA into HLA‑mismatched control cfDNA samples. We observed a limit of detection of 0.2% and a false positive rate of approximately 1 in 800,000 cfDNA molecules. We applied this platform to study 62 plasma samples banked from 18 lung transplant recipients with either biopsy-proven acute rejection, bronchiolitis obliterans syndrome (BOS) or no clinical rejection. For each clinical sample, we identified the fraction of donor‑derived cfDNA relative to total cfDNA. The level of donor-derived cfDNA was significantly elevated in patients diagnosed with acute rejection (10.30% ± 2.80%, n = 18), relative to patients with stable transplants (1.71% ± 0.50%, n = 24) or BOS (2.52% ± 0.62%, n = 20). In conclusion, we present a novel, validated application of ddPCR to non‑invasively assess acute lung allograft rejection using accurate quantification of donor‑derived cfDNA in primary clinical specimens. This technique should improve our ability to detect early acute rejection and improve long-term outcomes for lung transplant recipients.

Originally from Maryland, Andrew studied at Washington University in St. Louis for his undergraduate training in biology and computer science. After college, Andrew worked as a post-baccalaureate research fellow in the laboratory of Elliott Margulies at the National Human Genome Research Institute developing bioinformatic tools for eukaryotic genome assembly. Andrew subsequently returned to St. Louis to train as a physician scientist in the MD/PhD program at WashU. Andrew completed his PhD in the laboratory of Todd Druley, where he developed methods for error‑corrected DNA sequencing to study the role of rare clonal mutations in the development of leukemia and the clonal evolution of cells harboring leukemogenic mutations during physiological aging. Now in his last year of medical school, Andrew plans to continue training in internal medicine and hematology/oncology.


Droplet Digital™ PCR to Evaluate Systematic Gene Tagging with CRISPR/Cas9 to Illuminate Stem Cell Organization and Dynamics

Andrew Tucker
The Allen Institute for Cell Science

The Allen Institute for Cell Science is creating a dynamic visual model of hiPSC organization by generating a collection of fluorescently‑tagged clonal hiPSC lines. Our approach utilizes CRISPR/Cas9 gene editing to introduce fluorescent tags via homology driven repair (HDR) into genomic loci whose products localize to specific organelles. Editing yields hiPSC lines expressing fusion proteins unique to each cell line under endogenous regulation. Live cell imaging, image analysis and modeling, and open distribution to the scientific community of each unique cell line defines our endeavor. We present our gene editing protocol and discuss our unique screening and quality control pipeline. Using an RNP approach and FACS enrichment, we can select for potential on target HDR events. Upon clonal line generation, we perform several PCR‑based screens including Droplet Digital™ PCR (ddPCR™) to identify clones with precise on target fluorescent tag incorporation. Parallel ddPCR assays are used to quantify the genomic copy number of the tag and eliminate clones harboring imprecisely edited alleles containing the plasmid backbone. In experiments initiated to date, we have evaluated ~2000 clones and find that only ~25% of the clones are precisely edited. To date, we have implemented this ddPCR‑based screening approach to identify both monoallelic and bi‑allelic fluorescent tags incorporated into multiple genomic loci labeling ~15 major cellular structures including the actin and microtubule cytoskeleton, nuclear envelope, endoplasmic reticulum, and mitochondria (allencell.org).

Andrew graduated from the University of Toledo with a B.S. in Biology. He then moved to Seattle to work in the lab of Dr. Laura Crisa at the University of Washington, where he helped characterize the role of M2 polarized macrophages in Islet formation. Following this, he joined the lab of Dr. Michael Jensen at Seattle Children’s Research Institute where he worked to screen pathways that could be useful in boosting the effectiveness of Chimeric Antigen Receptor therapy in Acute Lymphoblastic Leukemia. He joined the Allen Institute in 2016 where as part of the Stem Cell and Gene Editing team he helped develop their gene‑editing and screening protocols for generating GFP knock‑in iPSC lines that are being used in an image based pipeline, with the goal of developing a fully integrated computer model of the cell.



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