Advances in stent-mediated gene delivery

1. Introduction

Atherosclerosis remains a leading cause of cardiovascular morbidity and mortality worldwide. Although phenomenal advances in stent designs and materials have improved outcomes of percutaneous coronary interventions, significant drawbacks remain that require attention. Gene-eluting stents (GES) hold the potential to modify the genes responsible for atherosclerosis and hence halt disease progression. In the last decade, we have seen significant improvement in the development of novel vectors that hold promise to increase gene transduction. Stents allow prolonged and localized gene elution and delivery to the site of injury. Several genes have been used in preclinical models with variable success to treat atherosclerosis and neointimal hyperplasia including nitric oxide synthase (NOS) isoforms, vascular endothelial growth factor (VEGF), and tissue inhibitor of metalloproteinases-1 (TIMP-1). The search for ideal vector and gene is in progress to address the epidemic of atherosclerosis. We believe that recent advances in stent designs, for example, drug-filled stents and bioresorbable stents can provide ideal delivery tools for GES and active collaboration should be established between academia and industry to develop to address gaps in the existing technology.

2. Atherosclerosis

Atherosclerosis is a chronic progressive inflammatory disease of the blood vessels that leads to ischemia of the myocardium, brain, and peripheral blood vessels and manifests as heart failure, stroke, and peripheral arterial disease. Together these represent a significant cause of cardiovascular morbidity and mortality globally and are responsible for enormous health-care costs. Advances in molecular cardiology have allowed recognition of multiple molecular pathways in the pathogenesis of atherosclerosis. Identification of genetic defects in the genesis of atherosclerosis allows the application of gene therapy.

Despite the advancements in drug-eluting coronary stents, current technology still falls far from the ideal solution. The fundamental issue is around the nonselective nature of cytostatic and cytotoxic drugs used in current stent technologies, including the scaffolds (bioresorbable stents). This delays re-endothelialization, which in turn increases the risk of late stent thrombosis; this necessitates the need for prolonged dual antiplatelet therapy, which is a significant concern for the elderly population. Furthermore, the scaffolds are in their infancy and require significant iterations to allow reliable scaffold delivery and predictable resorption post-stenting.

3. Gene-eluting stents

The field of cardiovascular gene therapy has seen limited clinical success. This is largely due to inefficient gene transduction the cause of which is multifactorial and includes systemic immune response to the vectors, insufficient delivery tools, and inadequate cell–vector interactions.[1] GES can overcome these limitations by using stents as delivery scaffolds for localized delivery of genes to the blood vessel wall and therefore allows prolonged elution and may also escape a systemic immune response. The potential cells that can be targeted in the blood vessel wall include endothelial cells, smooth muscle cells (SMCs), macrophages, and fibroblasts. Other noncellular targets include matrix metalloproteins and collagen.[2] To accomplish the desired clinical outcome, it is important that the vector can transverse the target cell and has adequate capacity to carry large DNA. The last decade has seen significant academic research on the concept of GES. Small and large preclinical models have been developed that show some promising results, but to date no human data is available on GES.

4. Vectors for cardiovascular gene therapy

An important consideration has been effective and safe delivery of gene to target lesion using vectors. Various approaches are employed, including coated and coatless techniques [3,4]; naked plasmids have shown ineffective gene transduction and efforts have been directed to increase plasmid stability to improve efficiency by adding liposomes and polymers. Although these strategies protect plasmids from early digestion and allow their increase of cellular uptake, these approaches lose cellular targeting. Our data showed a higher transfection of macrophages in the blood vessel wall when DNA liposomes [5] were used in a preclinical model. In another study, multilayer coating using protamine sulfate/plasmid DNA encoding hepatocyte growth factor for GES has shown successful gene delivery in preclinical model.[3]

There is renewed interest in nanocarriers for gene delivery. Poly(lactic-co-glycolic acid) (PLGA) and polyethyleneimine (PEI)-based nanocarriers have shown promise as effective delivery system because of their desirable properties such as effective gene transfection, better serum compatibility, and protection of drug from degradation.[6,7] PLGA showed better overall outcome as compared to PEI-based nanocarriers.[7] Limitations of PEI nanocarriers include low biodegradability; the contradiction between transfection efficiency and cytotoxicity while PLGA exhibits low release rate and low encapsulation efficiency of pDNA. Nonetheless, nanocarriers are exciting prospect for future of gene delivery.[8] In another study, Brito et al. demonstrated successful gene delivery using lipopolyplexes-embedded stents using precondensed plasmid DNA.[9] Similarly, Cohen-Sacks et al. showed gene delivery using poly-dl-lactide/glycolide and containing platelet-derived growth factor beta-receptor antisense in preclinical models.[10] Significant improvements in nanocarriers technology allow efficient gene transfection, less systemic toxicity, ease of modifications, and the promise of better clinical outcomes

Adenoviral (Ad) vectors are double-stranded (ds) DNA vectors that enter cell by endocytosis using coxsackie-adenovirus receptor. This vector (most serotypes) has shown effective transduction for both dividing and nondividing cells. The other advantage includes its capacity to carry larger DNA (30 kb). Although the transgene expression with this vector is robust especially in cardiovascular applications (e.g. heart failure), its widespread use is limited due to rapid clearance after T-cell immune response from residual adenoviral genes elimination of residual Ad genes (gutless vectors) has seen a reduced inflammatory response.[11] Furthermore, we have shown efficient SMC transduction in the blood vessel wall up to 28 days using GES.[5] In a study by Fishbein et al., coatless tethering of adenovirus gene vectors to stents using hydrolysable cross-linkers or polyallylamine bisphosphonate has shown promising results in cell culture.[12] Adeno-associated virus (AAV) are single-stranded (ss) DNA vectors that have more than 100 serotypes in nature. They induce much less inflammation and have shown good tropism for cardiovascular cells including cardiomyocytes, SMCs, and fibroblasts. AAV9 has shown significant promise for efficient gene transduction in preclinical models after intravenous injection.[13] We have shown persistent gene transduction in SMC with AAV2 up to 28 days in the preclinical model of GES.[14] AAV9 has not been used for GES and may be a superior vector for prolonged and persistent gene transduction of SMC. These distinct advantages have seen AAV as a preferred vector for cardiovascular system (CVS) gene therapy applications.

Lentiviral vectors are ssRNA vectors that can stably integrate into the host genome as cDNA via reverse transcription and therefore theoretically can produce prolonged and efficient gene transduction. However, to date, this vector has seen limited success for CVS application due to lack of tropism and concern for oncogenesis.[15]

5. Therapeutic genes for atherosclerosis

Several angiogenic factors have been explored for potential targets for GES including genes which by their overexpression prevent restenosis. Many genes have been evaluated in the bench-side and preclinical models. Retinoblastoma or the Rb gene and p21 gene have shown that mutated forms lead to overexpression and inhibition of vascular smooth muscle proliferation.[16]

Fibroblast growth factor (FGF) induces cell proliferation and production of proteases in endothelial cells. Intracoronary injection of Ad vectors encoding human FGF4 in a pig model of stress-induced MI resulted in improved myocardial perfusion and regional myocardial function.[17] However, to date, this angiogenic factor has not been used to target atherosclerosis via GES.

Enhanced expression of endothelial nitric oxide synthase (eNOS), which is a pleiotropic gene, has shown potential of reducing neointimal proliferation. Nitric oxide has potent vasodilator, antithrombotic, anti-inflammatory, and antiplatelet properties. Dysfunction in production or bioavailability of this protein leads to endothelial dysfunction.[18] We have shown that adenovirus-based eNOS delivery employing GES enhances re-endothelialization [5] and inhibits restenosis in preclinical model. However, in our other study, eNOS delivery using liposome–DNA complex did not reduce restenosis. This was related to nonspecific gene transfer to other cell lines, for example, macrophages. More recently, Brito et al. showed inhibition of restenosis following use of lipopolyplexes-eluted stent with eNOS expressing plasmid DNA in preclinical models. These differences can be explained by different formulations of liposomes used in these studies and highlights the fact that cell-specific interactions are crucial for targeting accurate cell lines.

Human VEGF-2 gene has also shown enhanced re-endothelialization and reduced neointimal hyperplasia in a preclinical model using plasmid-based GES.[4] Expression of VEGF leads to growth and recovery of vascular endothelium following injury. Prostacyclin synthase gene has also been shown to accelerate re-endothelialization and prevents neointimal formation in balloon-injured arteries in preclinical model; however, this effect was seen in conjunction with VEGF gene delivery and may very well be due to the later gene transduction.[19]

Similarly, in another study, tissue inhibitor of metalloproteinase-1 (TIMP-1) gene has shown to improve outcome in preventing restenosis in preclinical model using adenoviral delivery system.[20] Furthermore, Yin et al. showed targeting tissue factor pathway inhibitor gene using liposomes diminishes restenosis post stent insertion.[21] As atherosclerosis is an inflammatory disease, it is not beyond realization that we can target specific inflammatory pathways by either reducing adverse cytokines or enhancing cytokines which reduce inflammation, for example, interleukin 10.[22] IL-10 gene polymorphism has been shown to be associated with atherosclerosis. However, this preposition has been challenged recently by Sage et al. who found no association between absent IL10 and atherosclerosis in preclinical model. Finally, a hybrid approach using bilayered PLGA nanoparticles containing VEGF plasmid in the outer surface and paclitaxel in the inner core has been employed and showed that this approach resulted in endothelium healing while attenuates SMC proliferation.[23]

6. Expert opinion

Despite promising preclinical results of GES, significant hurdles still remain for its clinical application. First, a suitable nonimmunogenic vector (ideally a nonviral vector) with reliable elution and adequate cell targeting remains a significant challenge. Second, we need to ensure that appropriate genes are being transduced to achieve efficacy. Several therapeutic genes have been used to target native atherosclerosis and neointimal hyperplasia post-stenting. However, these small studies have shown mixed results and most promising results have been seen with NOS and VEGF isoforms. Another important practical hurdle is the requirement of a good manufacturing facility to ensure sterility and efficacy of the biological gene.

We believe that the progressive nature of atherosclerosis and changing demographics will see a significant health-care burden for both cardiovascular mortality and costs in the coming decades. GES can address atherosclerosis at molecular level and might help in halting its progression. Active collaboration between academia and medical device industry can overcome some of the remaining challenges for clinical translation of GES. Utilization of novel stent designs such as reservoir stent and drug-filled stents can provide an excellent platform for prolonged and efficient localized gene delivery. Another exciting avenue could be the employment of bioresorbable scaffolds as gene delivery tools. In the meantime, the pursuit for ideal vector and gene for GES application remains ongoing. Advances in stent-mediated gene delivery are summarized in Table 1.

Table 1. Advances in stent-mediated gene delivery.

Advances in gene delivery system	PEI- and PLGA-based nanocarriers [8]
Advances in target gene	-Tissue inhibitor of metalloproteinase-1 (TIMP-1) [20] -Tissue factor pathway inhibitor (TFPI) gene [21]
Advances in GES materials	-Hybrid stents, with VEGF plasmid in the outer surface and paclitaxel in the inner core [23] -Multilayer films for gene-eluting stent coating [3]
Advances in inhibition of restenosis	Lipopolyplexes-eluted stent with eNOS [9]

PLGA, poly(lactic-co-glycolic acid); PEI, polyethyleneimine; VEGF, vascular endothelial growth factor; eNOS, endothelial nitric oxide synthase.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.