Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork

Abstract

PurposeTo determine if proteasome inhibition using MG132 increased the efficiency of FIV vector–mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS).MethodsTM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 μM MG132 and transduced with FIV.GFP (green fluorescent protein)– or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 μM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA.ResultsIn the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 μM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus.ConclusionsProteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys

“…26 In the current study, LV-mediated Outflow resistance is produced by the TM, which is a targeting tissue for gene transfer to regulate IOP. 2,27 Efficient reporter gene delivery to the TM of monkey eyes in vivo 14,28-31 and ex vivo 12,[32][33][34] was achieved by vectors derived from adenovirus, 12,28,33,34 herpes simplex virus, 31 lentivirus, 29,30,32 and scAAVs. 14 Among these vectors, lentivirus has a relatively large packaging capacity and low immunogenicity.…”

Salt‐like transcription factor 4 promotes laryngeal cancer progression through transcriptional activation of ubiquitin‐specific protease 21 to stabilize Yin Yang 1

“…The above transfection sequences were designed and synthesized in GenePharma (Shanghai, China). Cells were treated with cycloheximide (CHX, 25 μg/ml, HY‐12320, MCE) or proteasome inhibitor MG132 (10 µM, HY‐13259, MCE), 20 followed by transfection with sh‐ USP21 .…”

Lentiviral Vectors for Ocular Gene Therapy

“…These observations strongly demonstrate the pertinence of such an approach. FIV vector transduction was improved in ex vivo macaca mulatta eye organ culture using a single injection of an MG132 proteasome inhibitor in the perfusion system one hour before the FIV injection [182]. MG132 final concentration in the anterior chamber was estimated to 20 µM.…”