Vigilant Vector: Heart-Specific Promoter in an Adeno-Associated Virus Vector for Cardioprotection

Abstract

Abstract-Repeated bouts of ischemia in the heart lead to fibrosis and eventually to heart failure. Although certain genes, such as SOD or hemoxygenase and antisense to AT 1 R, ACE, and (␤ 1 -AR can provide short-term protection of the heart from ischemia, there is no known mechanism for constantly responding to repeated incidences of ischemia. We hypothesized that a "vigilant vector," designed to be expressed specifically in the heart and switch on therapeutic genes only during hypoxia, would provide cardioprotection. To attain cardiac specificity, we inserted an MLC2v promoter into an adeno-associated virus (AAV) designed to deliver AS to AT 1 R and gfp. In in vitro experiments in cardiomyocytes (H9C2 cells), the MLC2v-AAV-gfp drove gene expression in all cells at levels comparable to a cytomegalovirus (CMV) promoter. In in vivo experiments, the rAAV-MLC2v-gfp was injected intravenously into mice or rats. Green fluoresence protein (GFP) DNA was located in kidney, heart (right and left ventricle), lung, adrenal and spleen. GFP mRNA, however, was expressed only in the heart and absent in other tissues. To switch on the rAAV transgene during ischemia, we inserted a hypoxia response element (HRE). This upregulates transcription when O 2 levels are low. Thus, there are 4 components to the vigilant vector; a gene switch (HRE), a heart-specific promoter (MLC2v), a therapeutic gene (AS-AT 1 R) and a reporter gene (gfp). To silence or lower basal level of expression while retaining specificity, we have reduced the length of the MLC2v promoter from 3 kb to 1775 bp or 281 bp. The truncated promoter is equally effective in heart specific expression. Preliminary studies with the rAAV-HRE-gfp in vitro show an increased expression in 1% O 2 in 4 to 6 hours. By adding additional hypoxia-inducible factor (HIF␣) (5 g), Key Words: ischemia Ⅲ cardiac function Ⅲ hypoxia Ⅲ myosin Ⅲ gene therapy Ⅲ adeno-associated virus T he human heart can be subject to repeated bouts of hypoxia, which leads to silent or overt myocardial tissue damage. 1 Cumulatively, this can lead to heart failure. In an attempt to combat this with gene therapy we are proposing the development of a "vigilant vector," inactive until switched on by hypoxia, that would protect the heart during ischemia with therapeutic genes. This concept requires the engineering of a stable vector that would contain 4 elements ( Figure 1): (1) a safe vector that could reach the heart by systemic injection and show stable expression of the gene in the heart; (2) a therapeutic gene for cardioprotection against ischemia; (3) a tissue-specific promoter to drive the transgene to express mRNA in the heart only; and (4) a gene switch that would switch on the tissue-specific promoter in response to hypoxia and that would switch off in response to normoxia.For the vector, the adeno-associated virus (AAV) is proving to be a stable, nonpathological vector. 2,3 There are several genes that could be considered for protection of the heart during ischemia. In a previous study 4 we had found that th...

AAV delivery of mineralocorticoid receptor shRNA prevents progression of cold-induced hypertension and attenuates renal damage

“…40,41 Nevertheless, a virus control (AAV.Control-shRNA) and a virus-free control (PBS) were used to exclude possibilities that may complicate interpretation of the experimental data due to the employment of viral vector. Control-shRNA is scrambled in sequence and has been proven, by BD Clontech (Palo Alto, CA, USA), not to interact with any known gene sequence.…”

Protection From Ischemic Heart Injury by a Vigilant Heme Oxygenase-1 Plasmid System

“…We have developed a strategy to use pathophysiological signals to switch on transgenes and amplify their expression, which we call "vigilant vector." 3,5 Recently, several studies have attempted to develop hypoxia regulated under the control of hypoxia response element promoters and have demonstrated that hypoxia response element promoters regulated vascular growth factor secretion in vitro and in MI animal models. However, this system is not ideal for treatment of ischemic heart disease because of the low efficiency of cardiac-specific gene expression in response to hypoxia loads.…”

“…3,4 In this study, we hypothesized that a vigilant HO-1 plasmid system can control the expression of HO-1 gene in ischemia myocardium and protect against ischemic injury in the heart.…”

In vivo imaging of mlc2v-luciferase, a cardiac-specific reporter gene expression in mice1

“…Previous studies have identified fragments of the promoter with 250 bp (15) or various other lengths (15,19,20). Those fragments seemed also to confer cardiac specific expression of reporter genes including Fluc and EGPF, which have been used to study gene regulations during cardiac development and differentiation (15,19,21).…”