Increased pressure for ex vivo transfection of antisense oligodeoxynucleotides in cardiac allografts improves transfection efficiency

Abstract

717 Introduction We have previously reported a novel DNA delivery system using pressure as a vector for ex vivo transfection of antisense oligodexoynucleotide (AS-ODN) in cardiac allografts. We hypothesized that increase of ex vivo pressure leads to an increase the efficiency of DNA delivery to myocardial cells without toxicity. Previous studies have shown that using AS-ODN to messenger RNA (mRNA) can block expression of proteins encoded by the mRNA. We have used ICAM-1 as a target for AS-ODN treatment since ICAM-1 has been shown to have an important role in reperfusion injury (RI), acute and chronic rejection. Methods ACI rats served as recipients of heterotopic PVG rat allografts. Donor hearts were perfused ex vivo with 1.3 ml of 80 μM flourescein labelled AS ODN and placed in a pressure chamber for 45 minutes, prior to transplantation. Hearts were procured 24 hours after transplantation. Efficiency of transfection was assessed by counting the number of nuclei labeled with flourescein AS ODN as a fraction of the total number of nuclei per field(Hoescht nuclei dye) using a Leica flouroscope. Function of the allograft was assessed by palpation and scored on a scale from 0 (no heart beat) to 4(strong heart beat). Toxicity was measured by percent wet weight (%WW) to assess cardiac edema, and myeloperoxidase (MPO) activity to assess neutrophil infiltration. Results Heterotopic allografts showed a significant increase in transfection with the increase of pressure applied to the donor heart: TableIncrease in pressure applied did not correlate with an increase in toxicity. Even at high pressure allograft function was uncompromised. Cardiac edema and neutrophil infiltration did not show any significant increase:TableConclusion We have shown that pressure can be a potent vector forex vivo transfecton of AS-ODN in cardiac allografts. Increase of pressure leads to significant improvement of transfection efficiency without causing toxic effects even at pressures as high as 8 atm. The use of donor allograft genetic manipulation may have a future role in the treatment of RI, acute and chronic rejection.