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D.F. Schorderet, V. Buchillier, C. Kostic, Y. Arsenijevic, F.L. Munier; Lentiviral-delivered Hammerhead Ribozyme Modulates the Allele-specific Expression of BIGH3 in HeLa Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):846.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the potential use of lentiviral-delivered hammerhead ribozyme in the modulation of an allele-specific BIGH3 mRNA. Methods: Mutations in specific regions of BIGH3 (TGFBI) that produce abnormal keratoepithelin are responsible for various corneal dystrophies, i.e. Groenouw type I, Lattice type I and IIIa, Avellino, Reis-Bucklers and Thiel-Behnke. The classical mutations occur at CpG dinucleotide not suitable for allele-specific hammerhead ribozyme recognition and catalysis. As a proof of principle, a ribozyme-accessible single nucleotide polymorphism located in BIGH3 that creates an allele-specific PstI site was identified in HeLa cells and a specific ribozyme was designed against that sequence. The ribozyme construct was placed under the control of the PGK1 promoter and inserted into a self-inactivating lentiviral vector. To increase the level of expression, the woodchuck hepatitis virus posstranscriptional regulatory element (WRE) was inserted downstream of the ribozyme sequence. Viruses were pseudotyped with the vesicular stomatitis virus glycoprotein (VSVG) and concentrated by ultracentrifugation. HeLa cells at a concentration of 104 cells/plate were infected with a lentivirus containing the ribozyme or the LacZ (control) construct and RNA was extracted 2.5 and 6.5 days after infection. RT-PCR was performed and amplimers were subcloned in pGEM plasmid. Sixty to 75 different clones were isolated from each experiment and digested with PstI in order to establish the frequency of each allele. Results: Transfection with the control lentivirus indicated that 80% and >95% of the cells were transfected and expressed the LacZ gene after 2.5 and 6.5 days, respectively. The ratio between the "mutated" and "normal" allele was 0.69 and 0.62 after 2.5 and 6.5 days, respectively. Conclusions: Lentiviral-mediated transfection of ribozymes is a selective although not very efficient way of modulating gene expression at the RNA level. Alternative strategies or the targeting of multiple sites may be needed to significantly reduce gene expression if this approach is to be used in clinics.
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