Brent A. French

Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors

BACKGROUND Efficient methods of introducing genes into myocardial cells must be developed before local somatic cell gene therapy can be implemented against myocardial disease. Although adenoviral (Ad5) vectors have been used to target rodent hearts and plasmid DNA has been directly injected into the myocardium of rats and dogs, the amounts of recombinant protein produced by these procedures have not been reported, and adenoviral vectors have not been used in large mammalian hearts. METHODS AND RESULTS Replication-deficient recombinant adenoviral vectors carrying either the luciferase or lacZ reporter genes were injected directly into the ventricular myocardium of adult domestic swine for evaluation of reporter gene expression. This procedure did not affect regional myocardial function as assessed by systolic wall thickening using ultrasonic crystals. Luciferase activity was detected 3 days after injection, increased markedly at 7 days, and then declined progressively at 14 and 21 days. Luciferase production was comparable in the right and left ventricular walls and increased with increasing amounts of virus, reaching 61 +/- 21 ng at the highest dose examined (3.6 x 10(9) plaque-forming units). The injection of 200 micrograms of plasmid DNA (pRSVL) produced levels of luciferase comparable to 1.8 x 10(8) plaque-forming units of recombinant Ad5; however, when normalized to the number of genes injected, the adenovirus was 140,000 times more efficient than plasmid DNA. Histochemical analysis of beta-galactosidase activity produced by a second Ad5 vector demonstrated that nearly all (> 95%) of the stained cells were cardiomyocytes and that the percentage of cardiomyocytes infected by the virus could be quite high in microscopic regions adjacent to the needle track (up to 75% in fields of 60 to 70 cells); however, Ad5-infected cells were rarely observed farther than 5 mm from the injection site. Furthermore, the Ad5 vector induced pronounced leukocytic infiltration that was far in excess of that seen after injection of vehicle alone. CONCLUSIONS This study demonstrates for the first time that direct intramyocardial injection of replication-deficient adenovirus can program recombinant gene expression in the cardiomyocytes of a large animal species with relevance to human physiology. The efficiency of adenovirus-mediated gene transfer is far superior to that of plasmid DNA injection, and this method appears to be capable of producing more recombinant protein. However, the cell-mediated immune response to the Ad5 vector and the limited distribution of reporter gene expression suggest that less immunogenic recombinant vectors and more homogeneous administration methods will be required before Ad5 vectors can be successfully used for phenotypic modulation.

High dose rate intracoronary radiation for inhibition of neointimal formation in the stented and balloon-injured porcine models of restenosis : Angiographic, morphometric, and histopathologic analyses

PURPOSE We examined the effects of intracoronary irradiation delivered at a high dose rate on neointimal hyperplasia after injury induced by two methods: balloon overstretch injury, and stent implantation in a porcine model of coronary restenosis. METHODS AND MATERIALS In 34 Hanford miniature swine, a segment of each coronary artery was targeted for injury and treatment. The artery segments were treated with 192Ir at doses of 10 Gy over 4 min (eight animals), 15 Gy over 6 min (nine animals), 25 Gy over 10 min (nine animals) or control (simulation wire only; eight animals). The treated segments were subjected to stent implantation (left anterior descending and right coronary artery) or balloon overstretch (circumflex) injury. Twenty-eight days later, repeat coronary angiography and sacrifice were done. Quantitative coronary angiography, morphometry, and extensive histopathologic analyses were carried out in a blinded fashion. RESULTS The change in minimal lumen diameter from postinjury to presacrifice in the stent-injured left anterior descending was -0.79 +/- 0.34 (mean: +/- SD) mm in the control group, compared to -0.43 +/- 0.35 mm in the 15 Gy (p = 0.04) and -0.21 +/- 0.50 mm in the 25 Gy (p = 0.01) groups; and in the balloon-injured circumflex was -0.31 +/- 0.22 mm in the control group compared to -0.03 +/- 0.18 mm in the 10 Gy (p = 0.05) and 0.00 +/- 0.33 in the 15 Gy (p = 0.01) groups. Percent area stenosis in the left anterior descending was 36 +/- 9% in the control group compared to 18 +/- 12% in the 15 Gy (p = 0.003) and 11 +/- 11% in the 25 Gy (p < 0.001) groups; and in the circumflex was 16 +/- 10% in the control groups, compared to 5 +/- 5% in the 15 Gy (p = 0.02) and 2 +/- 2% in the 25 Gy (p = 0.009) groups. Histopathology showed a striking reduction in the amount of neointima in the irradiated arteries compared with control vessels. Other radiation effects were stromal fibrin exudate, thinning of the media, and adventitial fibrosis and leukocyte infiltration in the radiated arterial segments. CONCLUSIONS High dose rate intracoronary irradiation with 192Ir effectively inhibits intimal proliferation after stent-induced as well as balloon-overstretch injury. This shorter treatment time (4 to 10 min) may provide a clinically practical approach to the prevention of restenosis after angioplasty.

Percutaneous transluminal in vivo gene transfer by recombinant adenovirus in normal porcine coronary arteries, atherosclerotic arteries, and two models of coronary restenosis.

BACKGROUND Gene therapy has been proposed as a possible solution to the problem of restenosis after coronary angioplasty. The current study was undertaken to assess conventional methods of gene transfer and to develop percutaneous techniques for introducing genes directly into the coronary arteries of large mammals. Since the anticipated targets of gene therapy against restenosis include atherosclerotic and previously instrumented arteries, we also evaluated gene transfer in atherosclerotic coronary arteries and in two porcine models of restenosis: one using intracoronary stents and a second using balloon overstretch angioplasty. METHODS AND RESULTS The conventional method of using perforated balloon catheters to deliver Lipofectin-DNA complexes directly into the coronary arteries of intact animals was applied to 18 porcine coronary arteries including normal arteries, hypercholesterolemic arteries, and those simulating restenosis. The results of this study were consistent with previously published results indicating that only low levels of luciferase gene expression could be obtained by Lipofectin-mediated gene transfer. We therefore undertook a second, parallel study to evaluate percutaneous transluminal in vivo gene transfer using a replication-deficient adenoviral vector. A comparison of the two studies revealed that the mean level of reporter gene expression in the cohort undergoing adenoviral infection was 100-fold higher than in the cohort undergoing Lipofection. Analysis of luciferase activity over time in normal arteries revealed that recombinant gene expression was half-maximal after 1 day, peaked within 1 week, was still half-maximal at 2 weeks, and declined to low levels by 4 weeks. Histochemical analysis of coronary arteries treated with a second adenovirus expressing a nuclear-localized beta-galactosidase gene demonstrated gene transfer to a limited number of cells in the media and adventitia. Immunohistochemical analysis of Ad5-infused arteries using a monoclonal antibody directed against CD44 identified a periadventitial infiltrate composed of leukocytes. CONCLUSIONS The recombinant adenoviral vectors proved to be far more effective than Lipofectin at delivering foreign genes directly into the coronary arteries of living mammals. Furthermore, the influences of hypercholesterolemia and arterial injury appeared to have little effect on the levels of gene expression obtained using either method. The results demonstrate that low-level recombinant gene expression, the major obstacle impeding gene therapy for the prevention of restenosis, can potentially be overcome by using adenoviral vectors to mediate coronary gene transfer in vivo. The duration of gene expression provided by these vectors and their effective deployment in atherosclerotic, balloon-overstretched, and stented coronary arteries suggest that recombinant adenovirus may have potential for evaluating gene therapy in the clinically informative porcine models of coronary restenosis.

The advent of adenovirus : gene therapy for cardiovascular disease

Although the era of gene therapy for human disease is relatively recent and brief to date, a succession of distinct phases can be recognized already by the rapidly expanding clinical horizons for correcting disease through the introduction of new genes.1,2 An initial period was focused on the strategy and expectation that hereditary disorders caused by the lack of a needed protein would be prevented by inserting normal copies of the gene into the germ line of affected patients or even unaffected heterozygotes who carried the mutation. In retrospect, although this assumption appeared logical enough at the time, daunting technical and ethical concerns soon were appreciated, such as the risks of inadvertent mutations caused by manipulation of germ line DNA, incomplete knowledge of the genetic sequences necessary to ensure accurate spatial and temporal expression of the curative gene, and inherent irreversibility of the approach. These limitations were compounded by the philosophical or social controversy concerning therapy directed at the germ line, which remains unresolved. Technical obstacles have also frustrated therapy directed at hematopoietic stem cells, the multipotential precursors of several lineages. Consequently, the field now is dominated by the contrasting paradigm of somatic cell gene therapy, the goal of modifying differentiated somatic cells with a therapeutic gene, not only for the single-gene disorders originally envisioned as the realm of this research but also for the acquired diseases encountered more commonly in clinical practice. Thus, gene therapy now must be envisioned as one potential way to deliver therapeutic proteins (or alter the expression of a patients own proteins) for, inherently, any disease. Given this goal of introducing foreign DNA to an appropriate organ in vivo, a number of generic methods exist, involving gene delivery by DNA alone, DNA in lipid particles (liposomes), or DNA incorporated into a suitable virus. The criteria that will ultimately define optimal approaches to gene therapy are centered on the interrelated issues of efficacy and safety, concerns that have brought replication-defective, recombinant adenoviruses to the forefront of gene therapy research. This Perspective will briefly explain the properties of adeno-

Fate of side branches after intracoronary implantation of the Gianturco-Roubin Flex-Stent for acute or threatened closure after percutaneous transluminal coronary angioplasty

Side branch occlusion may occur in the course of percutaneous transluminal coronary angioplasty (PTCA), particularly if complicated by site dissection. Concern that the additional placement of a stent may further jeopardize side branches is logical. Consequently, this study analyzed pre-PTCA, post-PTCA, poststent, and 6-month follow-up angiograms of 100 consecutive patients in whom 103 Gianturco-Roubin stents were implanted for acute or threatened closure after PTCA. Side branches were defined as major (> 50% of the stented vessel diameter) and minor (< 50%). Minor branches, often < 1 mm in diameter, were assessed only for patency. One hundred eight major branches, of which 33 were diseased (> 50% stenosis), and 129 minor branches were analyzed. Seven major branches (6%), all of which were diseased before PTCA, and 23 minor branches (18%) were lost after PTCA. Immediately after stent insertion, only 1 additional major and 1 minor branch were lost, whereas 2 of 7 major (29%) and 9 of 23 minor (39%) branches reappeared. At follow-up angiography, 7 major branches (6%) were more stenosed and 6 (6%) were improved compared with the angiogram before PTCA. Only 2 major (2%) and 5 minor (4%) branches remained occluded. Additionally, 2 major and 1 minor branch, which were patent after PTCA and stenting, were occluded at follow-up as a result of total occlusion of the stented segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter.

Recent studies have shown that two genes regulating myogenesis (MyoD and myogenin) are coexpressed with cardiac alpha-actin during early stages of skeletal muscle development. Myogenin and MyoD are members of a family of regulatory proteins which share a helix-loop-helix (HLH) motif required for dimerization and DNA binding. Myogenin and MyoD form heterodimers with the ubiquitous HLH protein E12 which bind cis-acting DNA elements that have an E box (CANNTG) at their core. E boxes are present in the control regions of numerous muscle-specific genes, although their functional importance in regulating many of these genes has not yet been evaluated. In this report we examine the possibility that myogenin (or MyoD) directly transactivates the cardiac alpha-actin promoter. Heterodimers of myogenin and E12 (or MyoD and E12) specifically bound a restriction fragment extending from -200 to -103 relative to the start of cardiac alpha-actin transcription. Methylation interference footprints pinpointed the site of interaction to an E box immediately adjacent to a previously identified CArG box (CArG3). Site-directed mutations to the DNA-binding site revealed that either an intact E box or an intact CArG3 is required for induction of the cardiac alpha-actin promoter in myoblasts and for transactivation by myogenin in cotransfected fibroblasts. However, deletion and substitution experiments indicate that the complex E box/CArG3 element alone does not confer muscle-specific expression to a minimal promoter. These results suggest that direct and indirect pathways involving multiple cis-acting elements mediate the induction of the cardiac alpha-actin promoter by myogenin and MyoD.

Use of nitric-oxide-eluting polymer-coated coronary stents for prevention of restenosis in pigs.

BackgroundRestenosis after angioplasty remains an unresolved problem despite an increase in use of coronary stents. It has been theorized that nitric oxide (NO) exerts several actions that can prevent restenosis. These include inhibition of proliferation of smooth muscle cells, prevention of arterial spasms, and decreasing aggregation of platelets in response to exposure to collagen. ObjectiveTo determine whether NO coated stents decrease restenosis in a pig balloon injury model. MethodsWe used coronary stents impregnated with a slow‐release precursor of NO in the porcine model of restenosis. Tantalum coil coronary stents (Cordis) were coated with a polymer impregnated with a slow‐release precursor of NO. Polymer‐coated stents without active precursors were used as controls. Oversized stents were mounted on a delivery balloon and subsequently deployed in the right coronary and left anterior descending arteries of each animal. ResultsRepeated recording of angiograms demonstrated that changes in minimum lumen diameter on going from immediately after stenting to 28‐day follow‐up for the control and NO‐eluting‐stent groups were similar, namely decreases of 1.89 ± 0.33 and 2.08 ± 0.28 mm, respectively. The morphometric results, showing that severe luminal narrowing occurred for both groups, were similar. The percentage area stenoses were 85 ± 5% for the control group and 84 ± 6% for the NO‐eluting group. Histology demonstrated that profuse formation of neointima and an inflammatory cell infiltrate occurred. ConclusionsSevere diameter stenosis occurred both for control and for treatment groups. The degree of angiographic stenosis was markedly worse than that previously reported for this model. Sustained release of a precursor of NO did not prevent restenosis in this model. This might have been due to a lack of efficacy of nitric oxide or to a profuse and overwhelming stimulatory effect of the polymer in the coated stents.