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JE Coleman, SL Semple-Rowland; Improvement in the Design and Production of a Lentiviral Vector System Significantly Increases Transduction Efficiency and Enhances its Potential for Use in Treatment of LCA1 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4618.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To improve the transduction efficiency of a lentiviral vector system and to confirm that the guanylate cyclase 1 (GC1) enzyme encoded by GC1 transgenes carried by lentivirus is functional. Methods: HIV1-derived, cPPT-CTS cis-elements were inserted upstream of the multiple cloning site of the lentivirus transducing vector (pTYF) to improve transduction efficiency of the virus, and viral titers were increased by optimizing the viral packaging and concentration procedures. VSVG-pseudotyped lentiviruses carrying the human EF1α and mouse IRBP1783 (mIRBP; provided by J. Boatright) promoters fused to placental alkaline phosphatase (PLAP), bovine GC1 or GC1-IRES-GFP (GC1/GFP) were produced. The transduction efficiencies and cell-specificities of these viruses were examined by injecting ∼105 TU of virus into the neural tubes of stage 10 chicken embryos. Expression of functional GC1 was examined using immunocytochemistry (ICC; antibody provided by A. Yamazaki) and activity assays performed on transiently transfected or virus-transduced cell cultures. Results: Examination of PLAP expression in retinal whole mounts and sections prepared from embryos injected with TYF-EF1α-PLAP virus revealed that PLAP was expressed in ≷90% of the cells and that these cells were distributed across all cell layers. Replacement of the EF1α promoter with mIRBP (TYF-mIRBP-PLAP) effectively restricted PLAP expression to the photoreceptor cells. GC1 protein was detected by ICC in TE671 cultures transfected with the pTYF-EF1α-GC1 plasmid vector and membrane preparations of transfected cells exhibited GCAP1-dependent GC activity in low calcium, indicating the presence of functional GC1. GC1 protein was also detected by ICC in photoreceptor cells in primary cultures of GUCY1*B (GC1-null) retina transfected with pTYF-mIRBP-GC1. TE671 cells infected with TYF-EF1α-GC1/GFP virus revealed that expression of GC1 was co-localized with GFP fluorescence, indicating stable transduction of the GC1 transgene. Conclusion: We demonstrate that modifications to our previous lentiviral vector system increased transduction efficiency from <20% to ≷90% in developing retina. The GC1 transgene used in our study encodes a functional GC1 enzyme and GC1 transgenes can be successfully transduced by lentivirus. Current studies are aimed at studying the efficacy of lentivirus-mediated GC1 gene therapy to treat retinal disease in the GUCY1*B chicken model of LCA1.
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