A number of clinical renal diseases are associated with and may be a result of disordered glomerular mesangial cell biology. Unlike the tubular components of the intact kidney, the rat renal glomerular mesangial cell is uniquely available to the circulation, is readily and reversibly provoked to enter mitosis, and is targetable by specific cell surface antibodies. Since there are a number of chronic and progressive renal diseases which are either acquired, idiopathic or have a genetic component, for which gene therapy, directed towards the glomerular mesangial cell, might prove useful. It is reasonable to target genetic expression vectors to mesangial cells in vivo with the anticipation of delivering genes whose products might alter the disordered mesangial cell biology associated with disease. We present background data indicating that the genetic vectors we propose using in vivo function within renal cells and renal mesangial cell in vitro. In addition, we present preliminary data to indicate that we have a candidate therapeutic gene to be delivered and that this gene profoundly affects the activated mesangial cell phenotype in the context of in vitro experiments.
The specific aims are therefore: 1. Determination of the optimum conditions for delivering and expressing reporter gene constructs in the renal mesangial cell in vivo by intra- arterial infusion of either a) retroviral preparations or of b) cationic immunoliposomes delivering episomally-replicating plasmid vectors. In initial studies, these vectors will contain constitutive rather than tissue-specific promoters in order to evaluate only whether a gene was delivered; to which cell(s) it was delivered; and the persistence of expression of the delivered gene. 2.Within the context of these vectors and methods of gene delivery, we will evaluate tissue-specific promoter whose in vitro properties suggest that they will target expression to an """"""""activated"""""""" rather that a quiescent mesangial cell. These promoters will include the smooth muscle a-actin promoter and the type IV collagenase promoter-both of which are known to be highly expressed in the activated mesangial cell but not in the quiescent cell. Each of these promoters has been cloned into expression vectors. 3. Substitution of a """"""""therapeutic"""""""" insert for a reporter gene. These will include antisense constructs directed towards mRNAs whose inhibition affects glomerular mesangial cell biology in vitro. At our current state knowledge, constructs expressing antisense RNAs to type IV collagenase and TGF-beta are likely candidates as target genes.
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