BIOE Seminar Series: Renee Cottle (Clemson University)

Friday, February 5, 2021
9:00 a.m.-10:00 a.m.
Virtual
Steven Jay
smjay@umd.edu

The Spring 2021 seminars will be held virtually on Fridays from 9:00 a.m. – 9:50 a.m., unless otherwise noted. All BIOE faculty, students, staff, postdocs, and affiliates as well as additional subscribers to our weekly seminars emails will receive Zoom event information the week of each seminar. 

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Renee Cottle, Ph.D. (Clemson University)

Assistant Professor of Biomedical Engineering

Ex vivo genome editing as a therapeutic approach for genetic diseases

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) system has proven to be the most promising gene editing tool available for therapeutic applications due to its facile design and robust targeting activity in human cells. When coupled with a donor template, CRISPR-Cas9 nucleases trigger the homologous directed repair pathway to precisely incorportate new gene sequences into the genome, which can be leveraged for correcting a disease-causing mutation or introducing a transgene in the patient’s cells. Despite the advantages of this approach, there are several barriers that impede its clinical application. One major hurdle is delivering a sufficient amount of CRISPR-Cas9 complexes and donor templates into target cell types. In previous research, we investigated microinjection, traditionally applied for in vitro fertilization, for direct, controlled delivery of nucleases and a donor template into human hematopoetic cells as a novel therapeutic strategy for sickle cell disease. We characterized a microinjection system, investigated the effects of microinjection on cell functionality, and demonstrated proof-of-principle of gene editing in human hematopoietic K562 cells microinjected with CRISPR-Cas9 along with a donor template. We found that injection negligibly affects the cell proliferation potential, provides high cell viability, and can be used to control the exposure of nucleases in injected cells. However, a major drawback of microinjection is the low throughput.

 

In constrast, nucleofection, a modified electroporation technique, is amenable for therapeutic applications. Currently, we are optimizing nucleofection of CRISPR-Cas9 nucleases with and without donor templates into hepatocytes as a therapeutic approach for inherited metabolic diseases of the liver. In particular, we are developing a curative strategy for hereditary tyrosinemia type I. Hereditary tyrosinemia type I is caused by loss-of-function mutations in the fumarylacetoacetate hydrolase (FAH) gene, which encodes an enzyme required for tyrosine catabolism, causing elevated levels of tyrosine in the blood and accumulation of toxic metabolites that initiate acute liver failure and increase the risk of hepatocellular carcinoma. One approach we are investigating involves introducing a frame shift mutation using CRISPR-Cas9 to permanently disrupt the 4-hydroxyphenylpyruvate dioxygenase (HPD) locus to turn off the tyrosine catabolism pathway upstream of the FAH enzyme and prevent formation of toxic metabolites in hepatocytes ex vivo. The gene modified hepatocytes would then be transplanted back into the patient to repopulate the liver with healthy hepatocytes. In preliminary results, we observed higher on-target activity in Hepa 1-6 cells nucleofected with Cas9 mRNA and ribonucleoproteins than to plasmid DNA with corresponding knockdown of Hpd. In primary hepatocytes, we observed high levels of on-target indels in Hpd of 60.2% with wild-type Cas9 mRNA, 70.3% with wild-type Cas9 ribonucleoproteins, and 61.5% with HiFi Cas9 ribonucleoproteins. We observed significantly higher cell viability and albumin production, up to 11-fold higher viability, in primary hepatocytes nucleofected with Cas9 ribonucleoproteins than with Cas9 mRNA. Further, we observed low cell viability in hepatocytes nucleofected with eGFP mRNA, which suggests that long synthetic mRNA is potentially more toxic than protein in hepatocytes. In ongoing work, we will evaluate CRISPR-Cas9 off-target indels in hepatocytes using next generation sequencing, and determine the extent that hepatocytes that have been gene modified ex vivo using CRISPR-Cas9 engraft and repopulate the liver and correct the disease phenotype in a Fah-deficient mouse model.

About the Speaker:

Dr. Renee Cottle is an Assistant Professor of Bioengineering at Clemson University. She earned her PhD in Biomedical Engineering from the Georgia Institute of Technology and Emory University in 2015. She completed a T32 Postdoctoral Fellowship in the Cardiovascular Research at Medical University of South Carolina in 2016 and started her faculty position at Clemson in August 2016. Her expertise is in gene editing, gene therapy, and nonviral delivery strategies. Dr. Cottle’s research group is focused on cell-based gene therapies for inherited metabolic diseases of the liver. Her research addresses technical barriers to advancing novel gene therapies for genetic disorders.

 


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