Successful DNA Transfer in Cultured Human Mesangial Cells Using Replication Deficient Recombinant Adenovirus

Norris Stanley Nahman, K. Reed Clark, Thomas J. Sferra, Kathleen E. Urban, Anne E. Troike, Julie Kronenberger, Daniel D. Sedmak

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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 (rAvβ-gal) containing a CMV/llacZ promoter-reporter expression cassette coding for β-galactosidase (β-gal). We assessed soluble and histologic β-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 rAvβ-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 rAvβ-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.

Original languageEnglish (US)
Pages (from-to)204-209
Number of pages6
JournalJournal of Investigative Medicine
Volume46
Issue number5
StatePublished - Jan 1 1998
Externally publishedYes

Fingerprint

Mesangial Cells
Adenoviridae
DNA
Fibronectins
Galactosidases
Gene transfer
Gene therapy
Biosynthesis
Transgenes
Genetic Therapy
Virion
Cytoplasm
Durability
Electrons
Phenotype

Keywords

  • Adenovirus
  • Gene therapy
  • Glomerulonephritis
  • Mesangial
  • Transduction

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Nahman, N. S., Clark, K. R., Sferra, T. J., Urban, K. E., Troike, A. E., Kronenberger, J., & Sedmak, D. D. (1998). Successful DNA Transfer in Cultured Human Mesangial Cells Using Replication Deficient Recombinant Adenovirus. Journal of Investigative Medicine, 46(5), 204-209.

Successful DNA Transfer in Cultured Human Mesangial Cells Using Replication Deficient Recombinant Adenovirus. / Nahman, Norris Stanley; Clark, K. Reed; Sferra, Thomas J.; Urban, Kathleen E.; Troike, Anne E.; Kronenberger, Julie; Sedmak, Daniel D.

In: Journal of Investigative Medicine, Vol. 46, No. 5, 01.01.1998, p. 204-209.

Research output: Contribution to journalArticle

Nahman, NS, Clark, KR, Sferra, TJ, Urban, KE, Troike, AE, Kronenberger, J & Sedmak, DD 1998, 'Successful DNA Transfer in Cultured Human Mesangial Cells Using Replication Deficient Recombinant Adenovirus', Journal of Investigative Medicine, vol. 46, no. 5, pp. 204-209.
Nahman, Norris Stanley ; Clark, K. Reed ; Sferra, Thomas J. ; Urban, Kathleen E. ; Troike, Anne E. ; Kronenberger, Julie ; Sedmak, Daniel D. / Successful DNA Transfer in Cultured Human Mesangial Cells Using Replication Deficient Recombinant Adenovirus. In: Journal of Investigative Medicine. 1998 ; Vol. 46, No. 5. pp. 204-209.
@article{7bf84c39135f4b16b96fe3e03412ef6a,
title = "Successful DNA Transfer in Cultured Human Mesangial Cells Using Replication Deficient Recombinant Adenovirus",
abstract = "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 (rAvβ-gal) containing a CMV/llacZ promoter-reporter expression cassette coding for β-galactosidase (β-gal). We assessed soluble and histologic β-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 rAvβ-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 rAvβ-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.",
keywords = "Adenovirus, Gene therapy, Glomerulonephritis, Mesangial, Transduction",
author = "Nahman, {Norris Stanley} and Clark, {K. Reed} and Sferra, {Thomas J.} and Urban, {Kathleen E.} and Troike, {Anne E.} and Julie Kronenberger and Sedmak, {Daniel D.}",
year = "1998",
month = "1",
day = "1",
language = "English (US)",
volume = "46",
pages = "204--209",
journal = "Journal of Investigative Medicine",
issn = "1081-5589",
publisher = "Lippincott Williams and Wilkins",
number = "5",

}

TY - JOUR

T1 - Successful DNA Transfer in Cultured Human Mesangial Cells Using Replication Deficient Recombinant Adenovirus

AU - Nahman, Norris Stanley

AU - Clark, K. Reed

AU - Sferra, Thomas J.

AU - Urban, Kathleen E.

AU - Troike, Anne E.

AU - Kronenberger, Julie

AU - Sedmak, Daniel D.

PY - 1998/1/1

Y1 - 1998/1/1

N2 - 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 (rAvβ-gal) containing a CMV/llacZ promoter-reporter expression cassette coding for β-galactosidase (β-gal). We assessed soluble and histologic β-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 rAvβ-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 rAvβ-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.

AB - 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 (rAvβ-gal) containing a CMV/llacZ promoter-reporter expression cassette coding for β-galactosidase (β-gal). We assessed soluble and histologic β-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 rAvβ-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 rAvβ-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.

KW - Adenovirus

KW - Gene therapy

KW - Glomerulonephritis

KW - Mesangial

KW - Transduction

UR - http://www.scopus.com/inward/record.url?scp=0032084707&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032084707&partnerID=8YFLogxK

M3 - Article

C2 - 9676052

AN - SCOPUS:0032084707

VL - 46

SP - 204

EP - 209

JO - Journal of Investigative Medicine

JF - Journal of Investigative Medicine

SN - 1081-5589

IS - 5

ER -