Use of the Rabbit Ear Artery to Serially Assess Foreign Protein Secretion After Site‐Specific Arterial Gene Transfer In Vivo: Evidence That Anatomic Identification of Successful Gene Transfer May Underestimate the Potential Magnitude of Transgene Expression

BackgroundThe development of molecular strategies for the treatment of restenosis has been hindered by low efficiencies of in vivo arterial transfection. Expression of intracellular marker proteins is generally evident in <1% of vascular smooth muscle cells after in vivo arterial transfection. Efforts to improve the efficiency of in vivo gene transfer have been further impeded by the use of transgenes encoding for intracellular marker proteins, necessitating tissue removal and limiting survey for expression to one point in time. Methods and ResultsTo study gene expression on a serial basis in vivo and determine the relation between a secreted gene product and transfection efficiency after in vivo arterial gene transfer, a method for performing and serially monitoring gene expression in vivo was developed using the central artery of the rabbit ear. Liposome-mediated transfection of plasmid DNA containing the gene for human growth hormone (hGH) was successfully performed in 18 of 23 arteries. Serum hGH levels measured 5 days after transfection ranged from 0.1 to 3.8 ng/mL (mean, 0.97 ng/mL); in contrast, serum drawn from the control arteries demonstrated no evidence of hGH proene duction. Serial measurement of hGH from transfected arteries demonstrated maximum hGH secretion 5 days after transfection and no detectable hormone after 20 days. Despite these levels of secreted gene product documented in vivo, immunohistochemical staining of sections taken from the rabbit ear artery at necropsy disclosed only rare cells in which there was evidence of successful transfection. ConclusionsThese experiments demonstrate a useful method of performing serial in vivo analyses of gene expression after vascular transfection and that anatomic analyses of transfection efficiency may underestimate the potential magnitude of expression in the case of a secreted gene product. These findings have implications for the clinical application of somatic gene therapy because low-efficiency transfection with a gene encoding for a secreted protein may achieve therapeutic effects not realized by transfection with genes encoding for proteins that remain intracellular.

[1]  R. Crystal,et al.  In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors. , 1993, Circulation research.

[2]  E. Nabel,et al.  Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo , 1993, Nature.

[3]  E. Nabel,et al.  Recombinant platelet-derived growth factor B gene expression in porcine arteries induce intimal hyperplasia in vivo. , 1993, The Journal of clinical investigation.

[4]  J. Isner,et al.  Percutaneous arterial gene transfer in a rabbit model. Efficiency in normal and balloon-dilated atherosclerotic arteries. , 1992, The Journal of clinical investigation.

[5]  E. Nabel,et al.  Transduction of a foreign histocompatibility gene into the arterial wall induces vasculitis. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Newman,et al.  Low Level In Vivo Gene Transfer Into the Arterial Wall Through a Perforated Balloon Catheter , 1992, Circulation.

[7]  J. Leiden,et al.  Systemic delivery of recombinant proteins by genetically modified myoblasts. , 1991, Science.

[8]  H. Blau,et al.  Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. , 1991, Science.

[9]  G. Acsadi,et al.  Conditions affecting direct gene transfer into rodent muscle in vivo. , 1991, BioTechniques.

[10]  J. Muhlestein,et al.  Direct in vivo gene transfer into the coronary and peripheral vasculatures of the intact dog. , 1991, Circulation.

[11]  B. Leibiger,et al.  Expression of exogenous DNA in rat liver cells after liposome-mediated transfection in vivo. , 1991, Biochemical and biophysical research communications.

[12]  E. Nabel,et al.  Introduction of vascular smooth muscle cells expressing recombinant genes in vivo. , 1991, Circulation.

[13]  E. Nabel,et al.  Site-specific gene expression in vivo by direct gene transfer into the arterial wall. , 1990, Science.

[14]  A. Brasier,et al.  Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. , 1989, BioTechniques.

[15]  I. Maxwell,et al.  Electroporation of mammalian cells with a firefly luciferase expression plasmid: kinetics of transient expression differ markedly among cell types. , 1988, DNA.

[16]  P. Russell,et al.  Implantation of genetically engineered fibroblasts into mice: implications for gene therapy. , 1987, Science.

[17]  A. Rogol,et al.  Impact of intensive venous sampling on characterization of pulsatile GH release. , 1987, The American journal of physiology.

[18]  K. Wood,et al.  Firefly luciferase gene: structure and expression in mammalian cells , 1987, Molecular and cellular biology.

[19]  D. Moore,et al.  Human growth hormone as a reporter gene in regulation studies employing transient gene expression , 1986, Molecular and cellular biology.