Abstract: TH-PO1094
Using CRISPR/Cas9 to Generate Second-Generation Cell Lines Used for Detecting Recurrent Focal Segmental Glomerulosclerosis (rFSGS) Patients
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Srivastava, Pankaj, Medical University of South Carolina, Charleston, South Carolina, United States
- Solanki, Ashish K., Medical University of South Carolina, Charleston, South Carolina, United States
- Arif, Ehtesham, Medical University of South Carolina, Charleston, South Carolina, United States
- Rahman, Bushra, Medical University of South Carolina, Charleston, South Carolina, United States
- Deng, Peifeng, Medical University of South Carolina, Charleston, South Carolina, United States
- Nihalani, Deepak, Medical University of South Carolina, Charleston, South Carolina, United States
Background
The gold standard for diagnosing glomerular diseases including FSGS requires invasive procedures such as renal biopsy, which may not be safe or feasible to perform in all patients. Recently, we published a cell-based assay for diagnosing rFSGS patients, which involved constructing cell lines using the rFSGS responsive genes BMF, IL-1β and IGFBP3. While the assay specifically diagnosed rFSGS pateints, the overall assay response was low (~1.5 fold increase over control). To boost the assay response and increase its specificity, a CRISPR/Cas9-based precise genome editing approach was employed, where highly specific and responsive second generation cell lines were created.
Methods
The Genome-CRISPR/Cas9 human AAVS1 Safe Harbor knock in kit-Puro (Cat# SH012) from GeneCopoeia, Inc. was used to individually knock in BMF, IL1-β or IGFBP3 gene promoter-Luciferase ORF’ cassette at the AAVS1 locus in HEK293 cells. PCR analyses were used to determine correct insertion of donor cassette at the AAVS1/safe harbor site. The response of constructed cell lines towards patient plasma from rFSGS and control patients was tested using a luciferase-based reporter assay.
Results
Remarkably, these cell lines showed significantly increased response to rFSGS patient plasma when compared to plasma from either control or non-rFSGS patients. An increase from ~1.5 fold (originally reported) to more than 10 fold was observed. Moreover, the response was consistent and showed more than 90% specificity in detecting rFSGS patients.
Conclusion
We present here the development and validation of a second-generation cell lines used for diagnosing rFSGS patients that are superior to conventional stably transfected cell lines. Unlike the first-generation cell lines, the second-generation cell lines have defined integration at the “safe harbor locus”. These cell lines are highly responsive and demonstrate low variability. Overall, our assay is noninvasive, highly sensitive and specific, and studies are being planned for conducting clinical trials to utilize its full diagnostic potential.
Funding
- NIDDK Support