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Juan Carabana, N. Comes, T. Borras; Developing a Pressure-inducible Gene Therapy Vector for the Treatment of Elevated Intraocular Pressure (IOP). Invest. Ophthalmol. Vis. Sci. 2011;52(14):4623.
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Our long-term goal is to develop inducible gene therapy vectors which will express a transgene under glaucomatous conditions. Our purpose here was to identify promoter sequences which would respond to elevated IOP and drive up the expression of a reporter gene. We selected the Matrix Gla (MGP) gene, whose expression we have shown to be highly upregulated by elevated IOP (HP) in the human trabecular meshwork (TM) tissue (1).
The MGP DNA promoter sequences (-748/+30) were amplified from pooled human genomic DNA (gDNA), sequence-verified and inserted into a promoterless secreted alkaline phosphatase (SEAP) reporter vector. The expression cassette (MGPpromoter-SEAP) was cloned into a pShuttle vector to generate a replication deficient Adenovirus (AdhMGP.SEAP). Human perfused anterior segments (n=2 pairs at submission) were perfused at 3-4 µl/min for 24h and then valve-injected with 20µl of the AdhMGP.SEAP high-titer stock. After infection, the flow of one eye was raised to achieve a ΔP of 30 mmHg, while the contralateral eye was left at baseline pressure (CP). Eyes were further perfused at their new constant P for 4 days (OD ΔP=30; OS ΔP=0). Effluents were collected every day for analysis of SEAP (chemiluminescence assay). At the end of the experiment, TM tissues were dissected and halved for RNA or DNA extraction to quantify SEAP transcripts and viral genomes by TaqMan PCR. Amplification values were normalized by 18S.cDNA and 18S.gDNA respectively. SEAP levels were normalized by the number of viral genomes in each TM.
SEAP was detected in all effluents at 48h and increased thereafter at each time point (2-20µg/ml), indicating that MGP sequences -748/+30 contain a strong promoter for expression in the human TM tissue. After correction for the number of viral genomes in each TM, the SEAP levels in the effluents from the eyes subjected to HP were 27.8X (pair #1) and 3.3X (pair #2) higher than in the CP control eye. Similarly, the SEAP transcripts present in the TM tissue at the end of experiment were 1.6X higher in the HP eye than in the CP control eye (both pairs).
In addition to containing promoter signaling sequences, the MGP -748/30 upstream-region seems to include regulatory elements that increase expression of the driven gene in the presence of HP. These pressure-responding regulatory elements function in the intact human perfused TM. Fusion of these sequences to candidate genes would allow controlled expression of therapeutic genes during a HP episode. This advance could help manage a future treatment of glaucoma by gene therapy.(1) Comes and Borras. Physiol Genomics 38,205(2009)
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