Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium.
We investigated the efficiency of activated polyamidoamine dendrimers, a new class of nonviral vectors, to transfect rabbit and human corneas in ex vivo culture. In addition to assessing the expression of a marker gene we have demonstrated that this approach can be used to induce the production of TNF receptor fusion protein (TNFR-Ig), a protein with therapeutic potential. Whole thickness rabbit or human corneas were transfected ex vivo with complexes consisting of dendrimers and plasmids containing lacZ or TNFR-Ig genes. Following optimisation 6-10% of the corneal endothelial cells expressed the marker gene. Expression was restricted to the endothelium and was maximal after transfection with 18:1 (w/w) activated dendrimer:plasmid DNA ratio and culture for 3 days. The supernatant of corneas transfected with TNFR-Ig plasmid contained TNFR-Ig protein which was able to inhibit TNF-mediated cytotoxicity in a bioassay. We have therefore shown that activated dendrimers are an efficient nonviral vector capable of transducing corneal endothelial cells ex vivo. They may have applications in gene-based approaches aimed at prevention of corneal allograft rejection or in treatment of other disorders of corneal endothelium.
Hudde
T
T
Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
Rayner
S A
SA
Comer
R M
RM
Weber
M
M
Isaacs
J D
JD
Waldmann
H
H
Larkin
D F
DF
George
A J
AJ
D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D046508:Culture Techniques; D004728:Endothelium, Corneal; D005822:Genetic Vectors / Q000008:administration & dosage*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007074:Immunoglobulin G; D011817:Rabbits; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics*; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D015703:Antigens, CD; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins