Abstract
Since insulin deficiency results from pancreatic beta-cell destruction, all type 1 and most type 2 diabetes patients eventually require life-long insulin injections. Insulin gene synthesis could also be impaired due to insulin gene mutations as observed in diabetic patients with MODY 10. At this point, insulin gene therapy could be very effective to recompense insulin deficiency under these circumstances. For this reason, an HIV-based lentiviral vector carrying the insulin gene under the control of insulin promoter (LentiINS) was generated, and its therapeutic efficacy was tested in a beta-cell transplant model lacking insulin produced by CRISPR/Cas9-mediated genetically engineered pancreatic beta cells. To generate an insulin knockout beta-cell transplant animal model of diabetes, a dual gene knockout plasmid system involving CRISPR/Cas9 was transfected into a mouse pancreatic beta cell line (Min6). Fluorescence microscopy and antibiotic selection were utilized to select the insulin gene knockout clones. Transplantation of the genetically engineered pancreatic beta cells under the kidney capsule of STZ-induced diabetic rats revealed LentiINS- but not LentiLacZ-infected Ins2KO cells transiently reduced hyperglycemia similar to that of MIN6 in diabetic animals. These results suggest LentiINS has the potential to functionally restore insulin production in an insulin knockout beta-cell transplant animal model of diabetes.
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Abbreviations
- Cas9:
-
CRISPR-associated system
- CRISPR:
-
Clustered regularly interspaced short palindromic repeat
- GFP:
-
Green fluorescent protein
- GLP-1:
-
Glucagon-like peptide-1
- HDR:
-
Homologous recombination repair
- KO:
-
Knockout
- MODY:
-
Maturity onset diabetes of the young
- PACAP:
-
Pituitary adenylate cyclase activating polypeptide
- RFP:
-
Red fluorescent protein
- T1D:
-
Type 1 diabetes
- T2D:
-
Type 2 diabetes
- VIP:
-
Vasoactive intestinal peptide
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Acknowledgments
This work was funded by Akdeniz University Scientific Research Administration Division (TDK-2018-2833) and the Scientific and Technological Research Council of Turkey (TUBITAK Grant No: 215S820).
Conflict of Interest Statement
No competing financial interests exist.
Author contributions:
Eksi YE performed the assays; Bisgin A collected and analyzed the data; Sanlioglu AD drafted the article; Balci MK, Azizoglu RO, and Griffith TS acted as consultants; Sanlioglu S designed the study. All co-authors have reviewed and approved the manuscript prior to submission.
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Eksi, Y.E. et al. (2022). Generation of a Beta-Cell Transplant Animal Model of Diabetes Using CRISPR Technology. In: Turksen, K. (eds) Cell Biology and Translational Medicine, Volume 18. Advances in Experimental Medicine and Biology(), vol 1409. Springer, Cham. https://doi.org/10.1007/5584_2022_746
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