Development of a gene therapy virus with a glucocorticoid-inducible MMP1 for the treatment of steroid glaucoma.
OBJECTIVE
To design a glucocorticoid-inducible virus vector overexpressing recombinant matrix metalloproteinase 1 (MMP1) and counteract extracellular matrix deposition in the trabecular meshwork only when steroid is present.


METHODS
Endogenous MMP1 expression was measured in primary human trabecular meshwork cells (HTM) treated with dexamethasone (DEX), triamcinolone acetate, and prednisolone acetate by TaqMan PCR. Wild-type and mutant MMP1 cDNAs were cloned downstream of a glucocorticoid response element (GRE) and P(TAL) promoter. Adenoviruses AdhGRE.MMP1 and AdhGRE.mutMMP1 were generated by homologous recombination. HTM cells and perfused human anterior segments were infected with the viruses, with and without DEX. MMP1 mRNA and protein were analyzed by TaqMan PCR, Western blot analysis, and ELISA. Activity of secreted MMP1 was evaluated by FRET and rat tail collagen type I assays. Immunohistochemistry was performed by double-labeling with anti-human MMP1 and collagen type I antibodies.


RESULTS
Endogenous MMP1 expression was greatly downregulated by the steroids. DEX-treated cells and perfused organ cultures infected with AdhGRE.MMP1 secreted high levels of MMP1. Induction of MMP1 cycled on and off with the addition or removal of DEX. Secreted wild-type MMP1 degraded collagen type I after activation, whereas secreted mutMMP1 did not. Immunohistochemistry showed faint staining of collagen type I in areas of trabecular meshwork with high MMP1 transgene expression.


CONCLUSIONS
The authors have developed a novel glucocorticoid-inducible adenovirus vector that overproduces MMP1 only in the presence of DEX. The availability of this vector sets up the foundation for the development of gene therapy drugs for the potential treatment of ocular hypertension in steroid-responsive patients.
Spiga
Maria-Grazia
MG
Department of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7041, USA.
Borrás
Teresa
T
D000256:Adenoviridae / Q000235:genetics*; D000293:Adolescent; D000368:Aged; D015153:Blotting, Western; D002478:Cells, Cultured; D002675:Child, Preschool; D003094:Collagen / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015316:Genetic Therapy; D005822:Genetic Vectors; D005902:Glaucoma, Open-Angle / Q000209:etiology / Q000628:therapy*; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D007429:Intraocular Pressure / Q000187:drug effects; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009924:Organ Culture Techniques; D011239:Prednisolone / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000201:enzymology / Q000821:virology; D014222:Triamcinolone Acetonide / Q000494:pharmacology; D055815:Young Adult
D005938:Glucocorticoids; D012333:RNA, Messenger; D003907:Dexamethasone; C009935:prednisolone acetate; D003094:Collagen; D011239:Prednisolone; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1; D014222:Triamcinolone Acetonide