Successful DNA transfer in cultured human mesangial cells using replication deficient recombinant adenovirus.
BACKGROUND
Efficient transfer of DNA into human mesangial cells is an essential first step in the development of gene therapies for mesangial cell-mediated glomerulopathies. In the present studies, we assessed the ability of replication deficient recombinant adenovirus to transfer DNA (transduce) into primary cultures of human mesangial cells.


METHODS
Primary cultures of human mesangial cells were transduced with an adenoviral vector (rAv beta-gal) containing a CMVllacZ promoter-reporter expression cassette coding for beta-galactosidase (beta-gal). We assessed soluble and histologic beta-gal activity, morphology, and phenotypic expression of mesangial cell transductants, durability of transduced mesangial cells by measuring transgene expression following trypsinization or after prolonged periods in culture and metabolic stability following transduction (as assessed by fibronectin biosynthesis).


RESULTS
We showed that rAv beta-gal efficiently transduced mesangial cells in a dose-dependent fashion at a multiplicity of infectious units (MOI) ranging from 1 to 400 plaque forming units/cell (pfu/cell). One hundred percent of mesangial cells were transduced at an MOI of 100 pfu/cell. By electron microscopic evaluation, viral particles of approximately 85-90 nm were demonstrated in the cytoplasm of transduced cells. Following transduction, legal levels rose rapidly and were 10-fold greater than baseline levels after 2 hours. Beta-gal levels continued to rise for 7 days following transduction. Transduction with rAv beta-gal was well tolerated; mesangial cell transductants maintained normal morphology and phenotype, tolerated 3 cycles of trypsinization and maintained normal constitutive production of fibronectin.


CONCLUSIONS
Gene transfer with adenovirus is an effective, well tolerated approach for introducing DNA into primary cultures of human mesangial cells.
Nahman
N S
NS
Department of Internal Medicine, Ohio State University, Columbus 43210, USA.
Clark
K R
KR
Sferra
T J
TJ
Urban
K E
KE
Troike
A E
AE
Kronenberger
J
J
Sedmak
D D
DD
D000256:Adenoviridae / Q000235:genetics*; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005920:Glomerular Mesangium / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007763:Lac Operon; D011995:Recombination, Genetic; D014779:Virus Replication