In vitro assessment for the efficacy of gene augmentation therapy using lentiviral vectors expressing ABCA4
Maryam Maleki Tehrani1 , Hamid Ahmadieh1 , Fatemeh Suri1 *
- Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences
Abstract: Inherited retinal diseases (IRDs) are the leading cause of blindness worldwide. Based on the results of genetic analysis on a cohort from a recently established national registry, defects in the ATP-binding cassette, sub-family A, and member 4 (ABCA4) gene are relatively common causes of IRDs among Iranian patients. The goal of this study is to develop a novel gene augmentation therapy using lentiviral vectors to target IRDs caused by mutations in the ABCA4 gene.
Methods: The subcloned pCLX-UBI-ABCA4-GFP vector was extracted from Stbl3-transformed bacteria using a maxi-plasmid extraction kit. To produce lentiviral particles, three lentiviral vectors, including the transfer vector containing the gene of interest (pCLX-UBI-ABCA4-GFP) and two helper vectors (psPAX2 and pCAG-VSVG), were used for the co-transfection of Lenti-X™ 293T cells. The resulting lentivirus was harvested and concentrated using the Takara Lenti-X™ Concentrator kit.
After confirming the efficacy of produced lentiviral particles in the transduction of control cells, retinal pigment epithelium (RPE) cells were isolated from a donated human eye globe and cultured in vitro. Upon confirming the identity of RPE cells using immunocytochemistry (ICC), these cells were transduced using concentrated virus particles. Flow cytometry analysis and real-time PCR (qPCR) were employed to assess green fluorescent protein (GFP) and ABCA4 expression levels in transduced RPE cells, respectively.
Results: Flow cytometry analysis of the transduced RPE cells showed that a considerable percentage of these cells were positive for GFP expression. Real-time PCR analysis demonstrated an increase in ABCA4 expression in the lentivirus-treated group compared to the non-treated control group at the in vitro level. These results indicated that the produced lentivirus worked properly.
Conclusion: The present study proposed a new gene augmentation approach to target IRDs caused by mutation at the ABCA4 gene. The findings of this investigation showed that delivering the targeted gene to retinal cells is highly successful. As a result, this system will be studied in the following stage using lab animals and in vivo settings.
