P237Targeted adeno-associated virus (AAV) vector for efficient gene transfer into endothelial cells in vivo

Background: Reduced activity and expression of sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a) is known to occur in HF. Method: Our 4-month study examined the effects of SERCA2a gene transfer in a swine volume-overload HF (VO-HF) model. Using Yorkshire-Landrace swine, HF was created by severing mitral apparatus chordae to induce mitral regurgitation. Results: At 2 months (M), a compensated state of VO-HF was found: prolongation of the rate of isovolumic relaxation (Tau), increased left ventricular internal diameter diastolic and systolic diameters (LVIDd, LVIDs). At 2M, intracoronary injection of adeno-associated virus serotype 1 vector carrying SERCA2a under a cytomegalovirus promoter (AAV1.SERCA2a) (n = 10) vs. saline (n = 6) was performed. At 4M, gene transfer resulted in (A) positive LV inotropic effects: (dP/dt)/P, 15.5 ± 3.0 sec − 1 SERCA2a-group vs. 21.2 ± 3.2 sec − 1 controls; p < 0.01; (B) a favorable trend in LV lusitropic effects: Tau, 0.037 ± 0.019 vs. 0.051 ± 0.01 msec, p = 0.09; (C) improvement in LV geometry: % change in LVIDs, +15 ± 11% controls vs. −3.0 ± 10% SERCA2a-group, p < 0.01. At 4M, BNP levels remained stable in post- SERCA2a gene transfer, in contrast to the progressive rising levels among controls. Further, cardiac SERCA2a expression was significantly decreased in controls whereas it was restored to normal levels in the SERCA2a group (Figure ). Lastly, there was no histopathological evidence of myocardial inflammatory reaction or necrosis. Conclusion: Overexpression of SERCA2a by in vivo AAV1-mediated intracoronary gene transfer preserved systolic function, potentially prevented diastolic dysfunction and improved ventricular remodeling.

Download Full-text

Variables Pertinent to the Efficiency of Adeno-Associated Virus (AAV) Vectors Mediated Gene Transfer to Human Vascular Endothelial Cells.

Hypertension Research ◽

10.1291/hypres.24.437 ◽

2001 ◽

Vol 24 (4) ◽

pp. 437-443 ◽

Cited By ~ 1

Author(s):

Shinji TERAMOTO ◽

Takeo ISHII ◽

Takeshi MATSUSE

Keyword(s):

Endothelial Cells ◽

Gene Transfer ◽

Vascular Endothelial Cells ◽

Vascular Endothelial ◽

Adeno Associated Virus

Download Full-text

Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction

Journal of Virology ◽

10.1128/jvi.74.8.3793-3803.2000 ◽

2000 ◽

Vol 74 (8) ◽

pp. 3793-3803 ◽

Cited By ~ 101

Author(s):

Carol H. Miao ◽

Hiroyuki Nakai ◽

Arthur R. Thompson ◽

Theresa A. Storm ◽

Winnie Chiu ◽

...

Keyword(s):

In Situ Hybridization ◽

Gene Transfer ◽

Genetic Diseases ◽

Critical Event ◽

Dimer Formation ◽

Adeno Associated Virus ◽

Virus Vectors ◽

Preclinical Trials

ABSTRACT Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about ∼5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only ∼5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5′-bromo-2′-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic β-galactosidase. Colabeling for β-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.

Download Full-text