Tissue-specific expression of an anti-proliferative hybrid transgene from the human smooth muscle alpha-actin promoter suppresses smooth muscle cell proliferation and neointima formation.
The retinoblastoma protein (Rb), a key regulator of cell cycle progression, can bind the transcription factor E2F converting it from a positive transcriptional factor capable of driving cells into S phase into a negative complex which arrests cells in G1. We have created a potent transcriptional repressor of E2F-dependent transcription by fusing the C-terminal fragment of Rb (p56) to the DNA and DP1-binding domains of E2F. Because the expression of E2F/56 fusion protein from a constitutive promoter was incompatible with virus growth, adenovirus constructs were prepared where transgenes were expressed from a fragment of the smooth muscle alpha-actin (SMA) promoter. Immunoblot and beta-galactosidase staining demonstrated smooth muscle-specific expression of this transcriptional element in vitro. The SMA-p56 and SMA-E2F/p56 adenoviral constructs also induced G0/G1 cell cycle arrest specifically in smooth muscle cells. Following administration to rat tissues, the SMA-beta-galactosidase construct exhibited expression in balloon-injured carotid arteries, but not in liver, bladder or skeletal muscle. Local delivery of the SMA-E2F/p56 adenoviral construct to balloon-injured carotid arteries inhibited intimal hyperplasia. Our results demonstrate that local delivery of the SMA-E2F/p56 adenoviral construct can limit intimal hyperplasia in balloon-injured vessels, while avoiding toxicity that could occur from the dissemination and expression of the viral transgene.
Wills
K N
KN
Canji, Inc., San Diego, CA, USA.
Mano
T
T
Avanzini
J B
JB
Nguyen
T
T
Antelman
D
D
Gregory
R J
RJ
Smith
R C
RC
Walsh
K
K
D000199:Actins / Q000235:genetics*; D000256:Adenoviridae / Q000235:genetics*; D000800:Angioplasty, Balloon / Q000009:adverse effects; D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods; D002339:Carotid Arteries / Q000473:pathology; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D006801:Humans; D006965:Hyperplasia / Q000517:prevention & control; D009131:Muscle, Smooth, Vascular / Q000473:pathology*; D051381:Rats; D016160:Retinoblastoma Protein / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes; D017539:Tunica Intima / Q000473:pathology; D001616:beta-Galactosidase / Q000235:genetics*
D000199:Actins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D001616:beta-Galactosidase