ABSTRACT
PURPOSE: We recently demonstrated that direct subretinal (SR) injection of adeno-associated virus (AAV) type 8 (AAV8) into photoreceptor cells and retinal pigment epithelium (RPE) is a highly efficient model of gene delivery. The current study compared transduction efficiency and expression patterns associated with various routes of vector administration. METHOD: The efficacy of intravitreal (VT), SR and subconjunctival (SC) injections for delivery of AAV8-derived vectors, i.e. those expressing luciferase (Luc) and enhanced green fluorescent protein (GFP) - AAV8/Luc and AAV8/GFP, respectively - were compared in an animal (mouse) model (n = 8 mice/group). Transduction efficiency and expression patterns were examined at post-injection weeks 1 and 2, and months 1, 3, 6 and 12 via in vivo imaging. RESULTS: One year after AAV injection, AAV8/Luc-treated mice exhibited stable and sustained high expression of vector in the VT and SR groups, but not in the SC group (VT:SR:SC = 3,218:2,923:115; 1 × 10(5 )photons/s). Histological analysis showed that GFP expression was observed in the inner retina of VT group mice, and in photoreceptor cells and RPE of SR group mice, whereas no GFP expression was noted in the SC group. Electroretinography (ERG) revealed adverse effects following SR delivery. CONCLUSIONS: Results suggest that both SR and VT injections of AAV8 vectors are useful routes for administering ocular gene therapy, and stress the importance of selecting an appropriate administration route, i.e. one that targets specific cells, for treating ocular disorders.
Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Intravitreal Injections/methods , Photoreceptor Cells, Vertebrate , Subretinal Fluid , Vitreous Body , Animals , Electroretinography , Eye Diseases/therapy , Genetic Therapy/adverse effects , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Humans , Intravitreal Injections/adverse effects , Luciferases/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Random Allocation , Transduction, Genetic/methodsABSTRACT
AIMS: In IgA nephropathy, circulating immune complexes containing IgA1 are deposited on the glomerular mesangium, causing mesangial cell proliferation and acceleration of extracellular matrix production. The suppressive effect of jacalin, a galactose-binding lectin, on IgA production in vitro was determined. MATERIALS & METHODS: Normal human peripheral blood mononuclear cells were stimulated with plate-bound anti-CD3 and Th2 stimulation, with or without jacalin. Regulatory and effector cell subsets were determined by flow cytometry, and immunoglobulin production by ELISA. RESULTS: Jacalin increased the ratio of CD4(+)CD25(+)CD152(+) Tregs:effector T cells in peripheral blood mononuclear cell cultures 60-fold. This CD4(+)CD25(+)CD152(+) Treg increase may have inhibited Th2-stimulated IgA production by B cells. CONCLUSION: Immune tolerance induced by jacalin can suppress IgA production.
Subject(s)
Glomerulonephritis, IGA/immunology , Immunoglobulin A/biosynthesis , Immunosuppressive Agents/pharmacology , Plant Lectins/pharmacology , T-Lymphocyte Subsets/drug effects , T-Lymphocytes, Regulatory/drug effects , Th2 Cells/drug effects , Antibody Formation/drug effects , Antigens, CD/immunology , Cell Separation , Cells, Cultured , Flow Cytometry , Humans , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Th2 Cells/immunologyABSTRACT
Alport syndrome is a hereditary nephropathy that results in irreversible, progressive renal failure. Recent reports suggested that bone marrow transplantation (BMT) has a beneficial, short-term effect on renal injury in Alport (Col4a3(-/-)) mice, but its long-term effects, especially with regard to survival, are unknown. In this study, Alport mice received a transplant of either wild-type or Col4a3(-/-) bone marrow cells. Surprising, laboratory evaluations and renal histology demonstrated similar findings in both transplanted groups. Transplanted cells accounted for >10% of glomerular cells at 8 wk, but type IV collagen alpha3 chains were not detected in glomerular basement membranes of either group by immunofluorescence or Western blot analysis, although Col4a3 mRNA in the kidney could be amplified by reverse transcription-PCR in knockout mice that received a transplant of wild-type bone marrow. Both transplanted groups, however, survived approximately 1.5 times longer than untreated knockout mice (log rank P < 0.05). These data suggested that irradiation, which preceded BMT, may have conferred a survival benefit; therefore, the survival time of knockout mice was assessed after sublethal irradiation (3, 6, and 7 Gy) without subsequent BMT. A strong positive correlation between irradiation dosage and survival time was identified (P < 0.0001). In conclusion, the improved survival observed in Alport mice that received a transplant of wild-type bone marrow might be primarily attributed to as-yet-unidentified effects of irradiation.
