Cambridge Healthtech Institute’s Ann Nguyen recently interviewed Jiing-Kuan Yee of City of Hope National Medical Center. Dr. Yee shares his keynote presentation on “Use Integration-Defective Lentiviral Vectors to Measure the Off-Target Effect of Gene Editing” at the Engineering Genes, Vectors, Constructs and Clones conference taking place January 18-19, 2016, as part of the 15th Annual PepTalk in San Diego, CA.
Q1: How did your research interests in pluripotent stem cells and disease treatment lead to your work improving algorithms for gene editing at City of Hope National Medical Center?
My major focus is on the use of pluripotent stem cells for disease treatment. With the conventional viral vector-based approach, we encounter two difficulties. First, random virus integration can cause insertional mutagenesis and increase the risk of adverse events in treated patients. Second, random vector integration subjects the expression of the therapeutic gene to position effect variegation caused by the host genomic DNA flanking the vector integration site. This problem can cause instability in the expression of the therapeutic gene. Gene editing allows precise alteration of the targeted genomic site and thus avoids the problems of insertional mutagenesis and position effect variegation.
Q2: Why use integration-defective lentiviral vectors (IDLV) to measure the off-target effect of gene editing (the focus of your keynote presentation)? Any challenges along with the benefits?
IDLV represents just one strategy of a number of approaches to measure potential off-target sites generated by gene editing nucleases. This strategy has no preset bias for measuring the off-target effect since the linear IDLV genome can be preferentially incorporated into any double strand DNA break in the host genome. The other advantage of IDLV is its ability to transduce many different cell types efficiently. Thus the strategy is not limited to only those cell lines that are easily transfectable. The challenge remains the sensitivity of this strategy to detect potential off-target effect. Our data clearly indicates the detection of the off-target effect above 1% but only sporadic detection between 0.5-1%. Whether an increase in the size of the transduced cell pool can increase the detection limit remains to be determined.
Q3: More broadly, what areas of interest in protein engineering will earn the most attention over the next few years?
In the case of gene editing, protein engineering to improve the specificity of nucleases including ZFN, TALEN and Cas9 to reduce potential off-target effect remains the priority to move the field forward.
Jiing-Kuan Yee, Ph.D., Professor, Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope National Medical Center
Prof. Yee received his BS in Biology from Fu Jen Catholic University, Taiwan. He completed his MS and Ph.D. in molecular biology from University of Texas, Dallas. He received his postdoctoral training in the Department of Pediatrics, University of California, San Diego. He then worked in Chiron Corp. for two years before joining the faculty at City of Hope National Medical Center. His research interests are to use pluripotent stem cells to model human diseases in vitro and to apply pluripotent stem cells in disease treatment.
To learn more about Dr. Yee’s presentation at the 8th Annual Engineering Genes, Vectors, Constructs and Clones conference, visit CHI-PepTalk.com/Genes-Vectors-Clones