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Imaging of Vascular Gene Therapy¹

¹ From the Department of Radiology, Johns Hopkins University School of Medicine, Traylor Bldg, Rm 330, 720 Rutland Ave, Baltimore, MD 21205. Received March 30, 2002; revision requested June 13; final revision received September 4; accepted September 30. Supported in part by National Institutes of Health grants R01 HL67195 and R01 HL66187. Address correspondence to the author (e-mail: xyang@mri.jhu.edu).

	ABSTRACT

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
Gene therapy is an exciting frontier in medicine today. Many genes have been shown to be useful for treatment of various vascular diseases, including chronic cardiac and limb ischemia syndromes, vasculoproliferative disorder, hypercholesterolemia, atherosclerosis, thrombosis, and hypertension. Precise delivery of genes into target vessels, efficient transfer of genes into vascular cells of the target, and prompt assessment of gene expression over time are three challenging tasks for successful vascular gene therapy. Thus, in vivo imaging methods that can be used to monitor gene delivery and localize gene expression are needed. Modern imaging techniques provide an opportunity to monitor and direct vascular gene therapy. Radiologists play a key role not only in developing and mastering endovascular genetic interventions but also in assessing the success of vascular gene therapy and directing further refinement of vascular gene therapy technology. This article provides an overview of the current status of imaging of vascular gene therapy.

© RSNA, 2003

Index terms: Arteries, MR, 9_*.12941², 9_*.12942 • Arteries, US, 9_*.12981, 9_*.12983, 9_*.12988 • Arteriosclerosis, 9_*.721 • Genes and genetics • Review

	INTRODUCTION

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
Atherosclerotic cardiovascular disease remains the leading cause of mortality in the United States (1). Gene therapy is a rapidly expanding field with great potential for the treatment of atherosclerotic cardiovascular diseases. Many genes have been shown preclinically to be useful for stimulating the growth of new blood vessels (angiogenesis) to improve cardiac and limb vascular insufficiency, for blocking postangioplasty or in-stent restenosis, for preventing acute thrombosis to avoid organ infarction, for stabilizing plaques to reduce plaque progression and the risk of plaque rupture, and for correcting the levels of low-density and high density lipoproteins to treat hypercholesterolemia (2–7).

Precise delivery of genes into targeted atherosclerotic plaques, efficient transfer of genes into endothelial cells and smooth muscle cells of the target, and prompt assessment of gene expression over time are three challenging tasks for successful vascular gene therapy. Catheter-based endovascular delivery of genes offers a promising therapeutic approach to the delivery of highly concentrated genetic materials and the minimization of undesirable systemic toxicity. Thus, interventional specialists should play an important role in delivering genetic materials to intended targets as vascular gene therapies become more widespread.

Our current knowledge about the biodistribution and/or in vivo pharmacokinetics of gene therapy relies mainly on results of in vitro studies, such as polymerase chain reaction, in situ hybridization, and immunohistochemical staining of tissues obtained at biopsy or autopsy. Hence, there is an urgent need for in vivo imaging methods capable of helping monitor gene delivery and localize gene expression. Of particular interest to our specialty, these in vivo imaging methods should enable radiologists to precisely visualize where the genes are delivered, how the genes interact with the target, and how long genes function.

Monitoring of vascular gene delivery and tracking of vascular gene expression are critical. After the initiation of vascular gene transfer, clinicians first must immediately assess the success of the primary gene therapy procedure and then promptly detect gene expression over time. An unrecognized failure of primary vascular gene delivery can delay treatment for weeks or months, while a proven failure of gene expression days after administration of genetic materials should indicate the need to promptly provide alternative treatment.

To date, most investigations about the imaging of gene therapy have focused primarily on noncardiovascular systems (8). The reasons for this revolve around the anatomic and physiologic characteristics of the cardiovascular system, specifically (a) thin vessel walls, which require high-resolution imaging modalities; (b) cardiac beating and vessel pulses, which require specific methods to reduce motion artifacts; (c) blood flow, which requires strategies to enhance the interaction between genes and vectors and the targeted vessel lesions; and (d) complicated endovascular interventional procedures. In recent encouraging attempts, different imaging modalities, such as magnetic resonance (MR) and optical imaging, as well as ultrasonography (US), have been tested for monitoring and guiding vascular gene delivery, tracking vascular gene expression, and enhancing vascular gene transfection and transduction. This article will provide an overview of the current status of imaging of vascular gene therapy.

	BASIC CONCEPT OF VASCULAR GENE THERAPY

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
A gene is an ordered sequence of nucleotides and is located at a specific position on a chromosome. Gene therapy is the introduction of recombinant genes into selective somatic cells to treat acquired or inherited disorders. Gene therapy is accomplished by using two primary therapeutic tactics: (a) gene augmentation, whereby the desired genes are provided or inserted into a patient’s genome without removal or alteration of the host cell’s original copy, and (b) gene blocking, whereby the desired genes are inserted to block the action of the endogenous gene (9).

Vascular Therapeutic Genes
Several genes have considerable potential for use in vascular therapy. For example, it is possible to treat thrombosis genetically by transfecting the arterial wall with different genes coding for therapeutic proteins such as thrombomodulin and hirudin (10,11). Restenosis is a common source of angioplasty and stent failure and is primarily caused by neointimal hyperplasia. Migration and proliferation of smooth muscle cells after mural injury is the principal event that leads to neointimal hyperplasia. A number of genes have been tested to block smooth muscle cell migration and proliferation (9,12). Currently, a vascular endothelial growth factor (VEGF) gene, which encodes for VEGF (a glucoprotein), is a promising gene for promoting angiogenesis and has been widely used in clinical trials (13,14). Table 1 summarizes the various types of gene-encoding products and the specific vascular problems for which they have been used. The reader is referred to several excellent reviews (7,9,15,16) for more detailed discussions about how genes affect atherosclerotic lesions.

In the early days of vascular gene therapy, many investigators were searching for ideal delivery vectors, including (a) viral vectors for gene transduction, such as retroviral (18), adenoviral (19), and adeno-associated viral vectors (20) and (b) nonviral vectors for gene transfection, such as DNA and RNA plasmids (21), synthetic oligonucleotides (22), and liposomes (23). However, each vector has its own advantages and disadvantages, and, none of these types of vectors have thus far been found to be ideal for both safe and efficient gene transfer and stable and sufficient gene expression. In an excellent cardiology-oriented review article (15), one group of investigators summarized the different vectors for vascular gene therapy in detail.

The primary advantage of the use of viral vectors for gene therapy is their high transduction rate. Adenoviruses are double-stranded linear DNA viruses that can transfer genes to both quiescent and dividing cells and do not integrate into the target cell chromosome. Retroviruses are single-stranded enveloped RNA viruses that can transfer by means of receptor-mediated endocytosis and integrate their genes into the chromosomes of targeted cells, a process restricted to dividing cells only. An adeno-associated virus is a defective human parvovirus, the genome of which is a single-stranded, linear, 5-kilobase DNA molecule (24). The common drawbacks for viral vectors include local and systemic toxicities, such as host inflammatory and immune responses directed against both viral and transgene proteins, and difficulties in manufacturing (15).

Nonviral vectors have a number of advantages as gene therapy vehicles, including easy construction, no need for an infectious agent, long-term transgene expression, and no immune responses. Nonviral vectors do not integrate with the host genome, thus reducing any possibility of mutagenesis. A plasmid is a circular double strand of DNA that is transcriptionally active and can replicate autonomously from the host cell (15). A liposome is a single or multilayered vehicle that can be synthesized to have a neutral, negative, or positive charge; can act as an artificial membrane; and can enter the cell by means of receptor-mediated endocytosis (9). Antisense oligonucleotides are short (10–30 base pairs in length) chemically synthesized DNA molecules that are designed to be complementary to the coding sequence of an RNA of interest (22). The primary disadvantage of these nonviral vectors is their low transfection efficiency in vivo (13,21).

Recent interest has been focused on the use of a new generation of lentivirus, a subclass of retroviruses, as a vector for transducing genes into vasculature (25–27). The rationale for choosing the lentivirus as the primary vector is that the lentiviral vector has several advantages: (a) the ability to integrate stably into the chromosomes of target cells, allowing reliable gene transfer and long-term gene expression; (b) no expression or need of viral genes for vector existence in host cells, thus avoiding immune responses to cells harboring virus-specific cytotoxic T cells; and (c) the ability to transduce both dividing and nondividing cells and, therefore, allow in vivo and in vitro gene transduction (28). It has been shown that the lentivirus has a higher transfer ability than nonviral vectors and a lower toxicity than adenovirus vectors. In vivo transduction of lentivirus has been performed in different animals, including mice, rats, rabbits, and pigs (26–29).

It is reasonable to consider an alternative to gene therapy, namely, simple delivery of an exogenous gene product (ie, a specific protein that is exogenously made by a gene) into the target without the use of vectors (9). However, this approach has some limitations, and its success depends directly on the characteristics of the exogenous proteins. First, these exogenous proteins with their specific secondary or tertiary structures are usually too large to pass easily through the cell membrane; thus, the delivery of these proteins is extremely difficult. In contrast, the vector-mediated gene transfer approach ensures delivery of a therapeutic gene into the target cells; thus, the gene-encoding protein remains intracellular to achieve a biologic effect (30). Second, although small-sized exogenous proteins can enter target cells, these proteins degrade rather quickly—in only a few minutes or hours—due to the presence of endogenous proteases in the cells. With a vector-mediated gene delivery approach, the intracellular promoter–driven process ensures the long-term expression of transgenes for days or months (15). In some cases, the transgenes can integrate into the genomes of the targeted cells, which results in permanent expression.

	APPROACHES TO VASCULAR GENE DELIVERY

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
The ability to efficiently deliver genes or vectors to the target vessel represents one of the major challenges to interventional specialists. Although systemic intravenous administration of gene or vectors is occasionally performed, the more common application approach is to use a local delivery technique. To date, various local gene delivery methods have been developed, including (a) ex vivo gene delivery systems, such as endovascular stents seeded with genetically modified endothelial cells that are then reimplanted into the target vessel (31); (b) surgery-based delivery, which involves direct injection of genes into surgically isolated target vessels (32); (c) percutaneous delivery, which involves direct administration of genes into the target through a percutaneous approach (33); and (d) catheter-based delivery (34). Of these gene delivery techniques, catheter-based gene delivery seems to hold the most promise for vascular applications in clinical practice.

The catheter-based gene delivery approach has some prominent advantages over other gene delivery methods, including (a) precise gene or vector delivery to a specific anatomic location, with transfection or transduction limited to the cells of interest; (b) minimal morbidity secondary to the delivery method; (c) no unwanted systemic effects; and (d) ability to be combined with existing conventional endovascular interventions, such as balloon angioplasty and stent placement. Since 1990, different delivery devices have been developed for endovascular local gene transfer (Table 2). These gene delivery catheters can be classified into three groups according to their working mechanisms: passive diffusion, pressure-driven diffusion, and mechanically or electrically enhanced delivery (Fig 1) (34). Different gene delivery devices from various companies each have their own advantages and disadvantages, however, and thus far none of the currently available gene delivery systems achieves all the necessary requirements for practical application (35).

View larger version (16K): [in this window] [in a new window]   Figure 1. Three types of endovascular local gene delivery devices. A, Passive diffusion device (Dispatch catheter; SciMed Life/Boston Scientific). B, Pressure-driven device (Remedy balloon catheter; SciMed Life/Boston Scientific). C, Mechanically enhanced device (Infiltrator; InterVentional Technologies/Boston Scientific). (Images courtesy of Mark Ungs.)

The Dispatch balloon (Boston Scientific) is an adaptation of the double-occlusion balloon catheter. This device creates multiple chambers within a vessel segment through a nonporous membrane that spans the distance between the limbs of an inflatable coil. A notable advantage of this system is that infusions can be prolonged without causing distal ischemia, because the Dispatch balloon is composed of an inner polyurethane sheath that allows unimpeded distal flow.

The disadvantage of passive-diffusion catheters is the low transfection rate, around 1.1%–16% (36). To efficiently transfer genes into smooth muscle cells in the media and adventitia, it is necessary to disrupt the physical barriers imposed by the continuous endothelium and the internal elastic lamina. This drawback motivated the subsequent development of pressure-driven balloon catheters and mechanically or electrically enhanced delivery catheters.

Pressure-driven Balloon Catheters
The concept of pressure-driven local delivery balloons began with the work of Wolinsky in the late 1980s, in which the idea of infusing a pharmacologically active compound locally to the vessel wall with a perforated balloon was described (37). Subsequently developed pressure-driven catheters have all been based on the principle of a balloon that can be inflated against the vessel wall and that contains variously sized pores or a gene-containing gel on the surface of the balloon. The Wolinsky balloon (perforated balloon) has multiple laser-drilled 25-µm-diameter micropores on its surface, and the gene or vector solution is delivered directly, by means of the orifice-mediated jet effect, through the micropores into the target vessel wall. Some investigators have shown that the perforated balloon is associated with low transfection efficiency, high prevalence of tissue injury, and poor control of delivery to cells of the target vessels (32,38,39).

The Crescendo catheter (Cordis) is a modified perforated balloon that has an outer membrane with 1,800 micropores that allow the gene or vector solution to "weep" gently onto the endothelium of the target vessel. The Remedy balloon catheter (double-channeled perfusion balloon; Boston Scientific) has a centrally located high-pressure angioplasty balloon that is surrounded by lower pressure multiple gene delivery channels consisting of 30–100-µm-diameter micropores on the surface. This type of balloon has features to minimize potential vascular injury from local gene delivery, including separate ports for balloon inflation and gene delivery and the mechanism of leakage of solutions through infusion pores instead of the use of a high-pressure jet stream as in the perforated balloon (40).

An infusion-sleeve catheter consists of an outer sleeve, which is loaded with the genes or vectors, and an inner balloon, which is used to inflate the sleeve against the arterial wall (41). The infusion-sleeve catheter does not cause important mural trauma (42,43).

The hydrogel-coated balloon is constructed with a sponge layer, a hydrophilic gel matrix 5–10 µm in thickness, on the surface of an angioplasty balloon (34). The sponge layer is first immersed or "painted" with the gene or vector solution. Then, inflation of the angioplasty balloon makes the gene-carrying sponge uniformly compress against the target vessel wall with a more even distribution of the genes or vectors (32,44). Some authors (15) believe that the hydrogel-coated balloon catheter is probably the most useful device for delivery of plasmid DNA vectors into the endothelium.

Mechanically or Electrically Enhanced Delivery Catheters
Mechanically enhanced delivery devices use physical means to penetrate the endothelium and the internal elastic lamina in order to target the deeper layers of the media and adventitia. An Infiltrator catheter (InterVentional Technologies) has a needle or microport strips that run lengthwise on a dilation balloon. Inflation of the balloon with a gene or vector solution results in penetration of the needle into the target vessel wall. Because of the mechanical penetration of the needle, the delivery efficiency of genes is high and washout by means of blood flow is slow (45).

An iontophoretic catheter adapts a flowing electric current to enhance the movement of vectors into the vessel wall (46). Recent efforts to develop a local vascular gene delivery system have focused on stent-based delivery approaches in which genetic materials are attached with nonresorbable polymer that is coated onto endovasuclar stainless steel or nitinol stents (47,48). Thus, mechanical opening of the gene-coating stents allows a long period of interaction between genes and target vessel wall.

	GENERAL CONCEPT OF IMAGING OF VASCULAR GENE THERAPY

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
The purpose of imaging vascular gene therapy is to judge the genetic management of cardiovascular disease. Radiologists can play a key role in determining the effectiveness of a given vascular gene therapy during or shortly after the therapy has been initiated, since phenotypic effects of gene therapy may not become apparent for weeks or months. Vascular gene therapy involves several components: a therapeutic gene, a vector to transfer that gene to an appropriate vascular smooth muscle cell or endothelial cell, and a method for delivery of this vector to a localized target vessel in vivo (15).

To focus on these components, imaging of vascular gene therapy must comprise four primary tasks: (a) imaging the genes themselves; (b) imaging gene-carrying vectors during gene or vector delivery; (c) imaging gene-encoded products (primarily proteins) after gene expression, which is referred to as imaging of the downstream products of gene therapy; and (d) imaging the clinical phenotypic consequences of vascular gene therapy (Table 3). At this point with current imaging technologies, it is not possible to detect a specific gene directly by using radiologic imaging methods. In addition, the in vivo transfection and transduction rates of vascular genes currently are very low. We can adapt different imaging means to assist in vivo gene delivery and expression. Thus, clinical imaging of vascular gene therapy should concentrate on three essential factors: (a) monitoring of the primary gene or vector delivery procedure to determine where genes are transferred, (b) enhancement of vascular gene transfection and transduction to determine how well genes interact with the target, and (c) tracking of vascular gene expression over time to assess how long the genes function at the target (Fig 2).

View larger version (30K): [in this window] [in a new window]   Figure 2. Clinical imaging of vascular gene therapy includes three components: monitoring of primary gene or vector delivery procedure (1), enhancement of vascular gene transfection and transduction (2), and tracking of vascular gene expression (3).

	MONITORING OF PRIMARY VASCULAR GENE OR VECTOR DELIVERY

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

MR Imaging
Cardiovascular MR imaging has some prominent advantages. It can be used to image the vessel wall, perform multiple diagnostic evaluations of organ function and morphology, and provide multiple image planes without the use of ionizing radiation. The authors of one study (27) have demonstrated the first encouraging evidence that catheter-based vascular gene delivery can be monitored in vivo with MR imaging. These authors produced a gadolinium–blue dye or gadolinium–gene vector medium by mixing gadopentetate dimeglumine (Magnevist; Berlex Laboratories, Montville, NJ) with either a blue dye or a third-generation lentiviral vector carrying a reporter gene, the green fluorescent protein (GFP) gene. In both media, gadolinium was used as an MR imaging marker to enable visualization of vessel wall enhancement, while the blue dye and GFP were used as tissue stain markers for histologic and immunohistochemical evaluation to help confirm the success of the transfer. The gadolinium–blue dye or gadolinium-GFP-lentivirus medium was delivered into the arteries of living animals using Remedy gene delivery catheters (Boston Scientific), and the entire primary delivery procedure was monitored with high-spatial-resolution MR imaging. The results of this study showed the potential of MR imaging to dynamically show where the gadolinium and genes are delivered, how the target portion is marked, and whether the gene transfer procedure causes complications such as perforation (Figs 3, 4). These investigators achieved a 100% correlation between gadolinium enhancement of vessel walls on MR images and blue dye and GFP staining of the corresponding tissues at histologic and immunohistochemical evaluation.

View larger version (99K): [in this window] [in a new window]   Figure 3. Gadolinium-blue dye transfer in pig femoral artery. A-F, Transverse high-spatial-resolution spin-echo MR images (repetition time msec/echo time msec, 150/10). A, Image obtained before gadolinium-blue dye infusion shows artery (open arrow) and vein (solid arrow). Scale bar = 1 mm. B-F, Images obtained at 3-minute intervals during gadolinium-dye infusion from minute 3 (B) to minute 15 (F). Arterial wall (solid arrow) shows only partial gadolinium enhancement at 2-4 o’clock position. Gadolinium chelate flows into adjacent tissue (open arrow) outside the target artery. G, H, Corresponding surgical photographs of control (G) and blue dye-targeted (H) arteries (solid arrows). Blue dye stains muscles (open arrow) outside the target artery. There is no such finding on the control side in G. I, Histologic section confirms that blue dye primarily stains a portion of artery wall at the 2-4 o’clock position, which is the location of wall dissection (arrow). (Original magnification, x20.) (Reprinted, with permission, from reference 27.)

View larger version (109K): [in this window] [in a new window]   Figure 4. Gadolinium-GFP-lentivirus transfer in pig iliac artery. A-F, Transverse high-spatial-resolution spin-echo MR images (150/10). A, Before gadolinium-GFP-lentivirus infusion, balloon is inflated in the artery (arrow) with 3% gadopentetate dimeglumine. V = vein, scale bar = 1 mm. B-F, Images obtained at 3-minute intervals during infusion from minute 3 (B) to minute 15 (F) show gadolinium enhancement of arterial wall via infusion channels (arrowheads) of gene delivery catheter. F, At minute 15, arterial wall is enhanced as a ring (arrow). G, H, Corresponding sections show immunohistochemical results in control (G) and GFP-targeted (H) arteries. GFP manifests as brown precipitate through all layers of intima (arrows), media, and adventitia. (Original magnification, x200.) (Reprinted, with permission, from reference 27.)

US Imaging
In the appropriate applications, US has some prominent advantages over other imaging modalities, including relative technical simplicity, portability, cost-effectiveness, capability to provide real-time imaging, and lack of radiation. US is used in a variety of cardiovascular applications. Recently, several US contrast agents or tracers, such as multilamellar liposomes, gas-filled bubbles, and fluorocarbon emulsions, have been developed. These agents and tracers are highly reflective compared to blood or tissue; they markedly enhance the echogenicity of the fluid or tissue in which they reside and thus greatly increase the sensitivity of US (50,51).

Authors of a recent study (49) demonstrated the possibility of using US to monitor primary vascular gene delivery. Figure 5 illustrates the principle of that study, in which a polymer-based echogenic microsphere was designed to function not only as a US contrast agent but also as a nonviral gene-carrying vector. The echogenic contrast agent gene-carrying vectors are made of biodegradable microspheres with a modal diameter of approximately 5 µm and are composed of poly(methylidene malonate 2.1.2) with a double-emulsion technique (52,53). Du et al (49) encapsulated GFP reporter gene plasmids into the microspheres and transferred these GFP microspheres into arterial walls in living animals. With US, the entire process of balloon positioning and inflation and deflation can be visualized in real time. The high-frequency US images obtained with an 8–15-MHz transducer enable clear differentiation of the structural details of the Remedy balloon (Fig 6). On transverse images obtained during infusion, microspheres delivered to the target vessel wall demonstrated a highly echogenic "starburst" appearance around the entire vessel wall, which correlated with immunohistochemical staining (Fig 6). In addition, color Doppler US allowed visualization of motion echo signals at the micropores of the gene infusion channels (caused by flow of microspheres from the gene infusion channels to the target vessel wall [Fig 6]) and displayed delivery-induced perforation in the target walls (Fig 7).

View larger version (22K): [in this window] [in a new window]   Figure 5. Principle of US imaging of microsphere-mediated vascular gene delivery. GFP gene plasmid is encapsulated into an echogenic biodegradable microsphere to form complexes. Then, GFP microspheres are delivered through a catheter into the arterial wall, which is monitored with high-frequency US. Thus, microspheres function not only as echogenic contrast agent but also as nonviral gene delivery vector.

View larger version (80K): [in this window] [in a new window]   Figure 6. A, Diagram of gene delivery balloon catheter. B, Corresponding longitudinal color Doppler US image (autoadjustable 8-15-MHz linear-array transducer) of inflated gene delivery balloon. Structural details of the device can be differentiated, including angioplasty balloon (), gene infusion channels (between open arrows), and guide wire (long solid arrow). Color signals show infused microsphere flow (short solid arrows) at balloon micropores. C, D, Comparison between US and immunohistochemically stained section of pig femoral artery. C, Transverse US image (autoadjustable 8-15-MHz linear-array transducer) shows that GFP microspheres flow out via balloon micropores, manifesting as a highly echogenic "starburst" ring evenly distributed around entire vessel wall. D, GFP expression confirms US findings by means of brown precipitate (arrows) surrounding entire target vessel wall. (Original magnification, x10.) E, F, Immunohistochemical results of untransfected (E) and GFP microsphere-transfected arteries (F). GFP manifests in F as brown precipitate observed from intima to the media. (Original magnification, x200.)

View larger version (81K): [in this window] [in a new window]   Figure 7. A-D, High-frequency color Doppler US images of catheter-based intravascular gene-microsphere delivery in pig femoral artery. Longitudinal (A) and transverse (B) images obtained before infusion show guide wire (arrowheads) in lumen of femoral artery (arrow). V = femoral vein. Longitudinal (C) and transverse (D) images obtained after infusion show hyperechoic arterial wall (open arrow) and perforation (solid arrows) of posterior wall, which was not observed on preinfusion images. E, Perforation (arrow) was confirmed at histologic examination. (Original magnification, x10.)

A US system with a transcutaneous high-frequency transducer provides excellent high-spatial-resolution images of the superficially located arteries such as the femoral and carotid arteries. However, such a system cannot create high-quality images of deeply located arteries such as the renal and iliac arteries and the aorta, owing to limited ultrasound penetration. To solve this problem, one may use an intravascular US system to help detect gene-carrying microspheres in the deeply located arteries.

	IMAGING-ENHANCED VASCULAR GENE DELIVERY AND EXPRESSION

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
Efficient gene transfer into a target-specific cell is another major challenge for vascular gene therapy. At this point, in vivo transfection and transduction rates of genes in vasculatures are very low, approximately 1% for nonviral vectors, for example (15). Recently, different strategies have been adopted to facilitate in vivo vascular gene transfection and transduction, with the development of imaging-enhanced vascular gene delivery and expression techniques and image-guided site-specific targeting of genes to vascular cells of interest.

Focused Ultrasound–enhanced Gene Delivery and Expression
To date, increasing evidence shows that focused ultrasound, applied with either a transcutaneous or an intravascular approach, can enhance nonviral vector-mediated vascular gene delivery and expression, and ultrasound-enhanced gene transfer may allow therapeutic levels to be achieved at safer doses when a virus is used as a gene-carrying vector. Several investigators (56–58) have demonstrated that ultrasound can enhance not only plasmid-mediated vascular gene delivery and expression up to 7.5–12-fold but also microbubble- or liposome-mediated vascular gene delivery and expression up to 10–300-fold (50,59–62). The latter may be based on the fact that the microbubble or liposome not only reflects sound waves but also absorbs sonic energy, which helps to enhance gene transfer and expression by the possible mechanisms of (a) ultrasonic heating and ultrasonic shock waves to the cell membrane, which induce cell-membrane porosity, reduce the thickness of the unstirred layer at the cell surface, and facilitate passage of microparticles across membranes (63,64); (b) ultrasonic cavitation (interaction between an ultrasonic field in liquid and microparticles within the isolated medium [65]), which ruptures microparticles and thereby increases gene release from microparticles at the target (60); (c) ultrasonic effects on cell regulation or transcription factors (59); and (d) local intraluminal infusion of microparticles, which results in prolonged retention (for up to 14 days) of genetic materials at the delivery site (66,67).

Authors of several studies (56,57,59,68) have also shown that low levels of ultrasound with intensities at 1–2 MHz appear to be sufficient to enhance gene delivery and expression. This can be incorporated into the clinical practice of US imaging. Further optimization of ultrasound parameters, such as ultrasonic intensity, frequency, exposure time, and duty cycle, is necessary to establish a standard guideline for the safe and efficient enhancement of vascular gene therapy by using ultrasound technology.

MR Imaging–enhanced Gene Delivery and Expression
Several investigators (64,69,70) have shown that gene transfection or expression can be substantially enhanced one- to fourfold with heating, as tested in different cells including prostate tumor cells, chondrocytes, and kidney cells. Moreover, incorporation of DNA with adjuvants or heat-sensitive promoters may further enhance gene transfection and expression with the use of heating (71–73). Proposed mechanisms for heat-enhancement of gene transfection include heating to fracture tissue, increasing the permeability of the plasma membrane and cell metabolism, and increasing the activity of heat-sensitive heat-shock proteins (71,73,74). In clinical practice, however, it is not feasible to heat the entire body of a patient. We need to generate local heat at the target site only. One of the strategies to deal with this is to have an internal heating source that is small enough to be placed easily into a local target via naturally existing anatomic channels, such as vessels.

One research group has developed an MR imaging heating guide wire (Fig 8). The design of this device is based on a coaxial antenna with an extended inner conductor (75,76). The heating guide wire has multiple functions: (a) receiver antenna to generate intravascular high-spatial-resolution MR images of atherosclerotic plaques of the vessel wall (77,78); (b) conventional guide wire to guide endovascular interventions with MR imaging (79,80); and (c) intravascular heating source to deliver external thermal energy into the target vessel wall during MR imaging of vascular gene delivery and thereby enhance vascular gene transfection (76). By delivering thermal energy with this "three-in-one" device from an external 2.45-GHz microwave generator, the investigators (81) have achieved a three- to fourfold increase in GFP-lentiviral transduction in a series of in vitro studies with vascular smooth muscle cell phantoms (Fig 9).

View larger version (127K): [in this window] [in a new window]   Figure 8. A, A 0.032-inch MR imaging heating guide wire (MRIHG; Surgi-Vision, Gaithersburg, Md), the proximal end of which is connected to a matching-tuning decoupling circuit (arrow). B, Diagram shows in vivo experimental setup in rabbit aorta. MR imaging heating guide wire is placed within a balloon that is positioned side by side with a fiberoptic sensor in rabbit aorta. Temperature increase during intravascular heating by the guide wire-microwave generator (MWG) system is recorded by the fiberoptic sensor connected to a thermometer. C, D, Transverse high-spatial-resolution MR images of rabbit aorta with MR imaging heating guide wire during balloon inflation. Aortic wall (arrows) is clearly depicted on T1-weighted spin-echo image in C (500/14, 4 x 4-cm field of view, 256 x 256 matrix) and T2-weighted fast spin-echo image in D (2,000/100, 8 x 8-cm field of view, 256 x 256 matrix). E, Sagittal T2-weighted fast spin-echo MR image (2,000/100, 6 x 6-cm field of view, 256 x 256 matrix) of balloon inflated with saline shows the metal marks (arrows) of the balloon.

View larger version (80K): [in this window] [in a new window]   Figure 9. A, In an in vitro experimental setup using a four-chamber cell-culture slide, the MR imaging heating guide wire (arrow) is positioned beneath chamber 4. B, Graph shows that temperature increases at bottoms of four-chamber slide during heating by the guide-wire system with 2.54-MHz microwave energy. C, Graph shows corresponding GFP-lentiviral transduction rates in each chamber. D, Corresponding confocal microscopic images in chambers 1-4. Smooth muscle cell nuclei are stained with blue dye, while expressed GFPs manifest as green light. Temperature increase and transduction rate are highest for chamber 4, which is directly heated by the guide-wire-microwave system.

	IMAGING FOR SITE-SPECIFIC TARGETING OF GENES AND VECTORS

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

US of Site-specific Targeting
Site-targeted US contrast agents that use liposomes or microbubbles are designed for specific and sensitive enhancement of the acoustic reflectivity of pathologic tissue that would otherwise be difficult to distinguish from surrounding normal tissue (82). Echogenic liposomes are attractive as targetable contrast agents, because their bilayer phospholipids can be used for coupling molecules (eg, by means of avidin-biotin binding) to the external portion of the liposomes, thereby facilitating site-specific targeting (83). Depending on the ligands conjugated to the liposomes, these US contrast agents also have the advantage of targeting different components of atherosclerosis, such as proliferating smooth muscle cells, fibrin and fibrinogen, and the extracellular matrix (including fibronectin, laminin, and collagen) (84). In addition, surface-modified microbubbles or liposomes, with their small size, can enhance arterial uptake due to the change in microbubble or liposome surface charge (85,86).

An example of US-based site-specific targeting for vascular use is acoustic microscopy (87). This technique has been tested primarily for targeting of fibrin in the thrombi of fibrous plaques by using antibody-conjugated echogenic liposomes and emulsions (82,83). To learn more about US-based site-specific targeting, the reader is referred to an excellent pharmacology-oriented review article (88).

MR Imaging of Site-specific Targeting
Recent investigations (89) have resulted in the development of a fibrin-targeted, paramagnetic, nanoparticulate MR contrast agent that allows enhanced sensitive detection and quantification of occult microthrombi within the intimal surface of atherosclerotic vessels. This agent is a ligand-directed, lipid-encapsulated, liquid perfluorocarbon nanoparticle (250-nm diameter); it has a prolonged systemic half-life and can carry high gadolinium-based–agent payloads. The results of these studies suggest that MR imaging of fibrin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of fibrin and may allow early direct identification of vulnerable plaques, which would lead to timely therapeutic decisions. Other investigators (90) have also introduced a site-targeted MR contrast agent by conjugating a C2-glutathione S-transferase fusion protein to superparamagnetic iron oxide nanoparticles, which allows noninvasive detection of apoptosis at MR imaging. The above-mentioned site-specific US liposomes and microbubbles and paramagnetic nanospheres can be combined with microparticle-mediated vascular gene therapy (91).

	TRACKING VASCULAR GENE EXPRESSION

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
Tracking of gene expression means using imaging methods to assess gene function by detecting functional transgene-encoding proteins (referred to as imaging downstream) at the targets over time.

Tracking Exogenous Marker Gene Expression
Different exogenous imaging marker genes, also termed imaging reporter genes, have been developed. The currently available imaging marker genes have been listed in an excellent radiology-oriented review article (8). On the basis of their working mechanisms, these imaging marker genes can be divided into two groups: (a) marker genes encoding for intracellular proteins (such as enzymes) to modify imaging prodrugs, so that tissue accumulation of such drugs reflects the gene expression, and (b) marker genes encoding for cell-surface ligand-binding proteins or peptides (such as receptors) that can then be targeted by using corresponding imaging tracers (92).

To date, various imaging modalities, including nuclear and optical imaging, as well as MR imaging, have been used to detect expression of these imaging marker genes (93–95). Figure 10 presents some typical examples of the detection of imaging marker genes with different imaging modalities through various pathways. An example of a marker gene for nuclear imaging is the herpes simplex virus 1 thymidine kinase (HSV-tk) gene, which encodes an intracellular enzyme, thymidine kinase. Thymidine kinase can phosphorylate different radiolabeled substrates such as iodine 131–FIAU (5-iodo-2'-deoxy-1-ß-D-arabinofuranosyl-5-iodo-uracil), which are then "trapped" in the HSV-tk–targeted cells. Thus, cellular retention of radioactivity is an indicator of thymidine kinase gene expression under nuclear imaging (96).

View larger version (27K): [in this window] [in a new window]   Figure 10. Typical examples of imaging modalities used to track imaging marker gene expression by directly demonstrating downstream gene-encoding products through various pathways. HSV-tk = herpes simplex virus 1 thymidine kinase.

In fluorescent approaches, an external source of light is required for excitation of the fluorescent proteins (ie, GFP and RFP), while in bioluminescent approaches the reporter proteins (eg, luciferase) produce light by using appropriate substrates. Luciferases are a family of photoproteins that can be isolated from a large variety of insects, marine organisms, and prokaryotes (100). Molecular oxygen reacts with luciferase and luciferin, resulting in the formation of a luciferase-bound peroxyluciferin intermediate, which releases photons of visible light with emission spectra between 400 and 620 nm (100,101).

The emergence of the viable marker GFP has opened the door for the convenient use of intact living cells and organisms as experimental systems in fields ranging from cell biology to biomedicine (102–105). GFP, which fluoresces autonomously in nearly all cells that have been tested, is increasingly being used as a convenient and sensitive reporter of transgene expression that can be detected at fluorescence microscopy or flow cytometry (102,106). Any of the vectors designed for gene expression can be used to make constructs to carry and express GFP in different cells or organisms, either alone or as a fusion protein. The main advantage of GFP as an imaging marker is its high quantum yield (107), but its emission spectra intersect with the spectra of endogenous fluorophores such as collagen, leading to greater autofluorescence (108). In contrast, RFP excitation and emission maxima occur at longer wavelengths, resulting in less autofluorescence and deeper penetration through tissue; however, the quantum yield of RFP is low at this point (107,108).

Recently, optical imaging has been tested to track in vivo GFP and RFP gene expression in vasculatures (26,109,110). Although GFP and RFP genes have been successfully transfected in different animals such as mice, rabbits, and pigs with no clinical manifestations of immunoreactions after transfection, at this point optical imaging of GFP- and RFP-based vascular gene expression can be considered only as a laboratory imaging tool for in vivo testing and refining of vascular gene therapy technology.

Optical Imaging of Vascular Gene Expression
Medical optical imaging is used to detect light signal transmission, reflection, or emission from scattering media such as tissues, to determine interior structure and chemical content. Recently, investigators (26,109) have attempted to extend in vitro fluorescence microscopic imaging of GFP expressed from vascular specimens to in vivo digital optical imaging of GFP expressed from vessel walls in living animals. First, by testing different retroviral vectors that express an enhanced-GFP gene, some investigators (103,104) established a modified enhanced-GFP–lentiviral reporter system. With this system, GFP detection was improved up to 100-fold over that achieved previously. Subsequently, investigators (26,109,111) using a surgery- or catheter-based approach transduced GFP-lentiviral vectors into the carotid or femoral arteries of rabbits and pigs (Fig 11) and then used a digital optical imaging system to detect fluorescent signals emitted from GFP. In vivo optical imaging exhibited areas of high signal intensity that are representative of fluorophore emission in GFP-targeted arteries, while there was no such enhancement in untransduced control arteries (Fig 12). These optical imaging findings were confirmed with fluorescent microscopy, confocal microscopy, or immunohistochemical evaluation. This technical development provides an in vivo optical imaging method to localize discrete areas of fluorescent gene expression and to track the level and duration of fluorescent gene expression in vasculatures.

View larger version (73K): [in this window] [in a new window]   Figure 11. Surgery-based gene delivery into rabbit carotid artery (solid arrow). GFP-lentiviral solution (open arrow) is injected after isolation of the artery by means of two Sentinel loops (Sherwood Medical; St Louis, Mo) (arrowheads).

View larger version (87K): [in this window] [in a new window]   Figure 12. A-C, Non-GFP-targeted rabbit carotid artery. D-F, GFP-targeted rabbit carotid artery. A, D, On digital optical images, GFP-targeted tissue in D shows fluorescent signal enhancement, while non-GFP-targeted tissue in A shows no such enhancement. Scale bar = 1 mm. B, E, Corresponding images from fluorescent microscopy show strong green fluorescence of endothelium and internal elastic lamina (arrows) in targeted tissue in E but not in nontargeted tissue in B, although elastic lamina and endothelium in B show some autofluorescence. (Original magnification, x200.) C, F, At immunohistochemical evaluation, brown precipitate (arrowheads) in target tissue in F confirms findings shown in D and E. (Anti-GFP antibody and diaminobenzidine substrate stain; original magnification, x200.)

	COMBINING IMAGING MARKERS AND VASCULAR GENE THERAPY

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
To date, radiologists have developed different methods for in vivo detection of imaging marker gene expression in nonvascular and vascular systems. Some of the existing imaging marker genes encode proteins such as thymidine kinase and tyrosinase, which have certain anticancer and antiviral effects when linked to therapeutic prodrugs such as thymidine kinase–converted ganciclovir (113). However, other imaging marker gene–encoded proteins, such as GFP and transferrin receptors, function only as imaging markers with no therapeutic effects.

It now seems appropriate to combine newly developed techniques for the detection of imaging marker gene expression with vascular genetic therapies to treat atherosclerotic lesions. One strategy to address this is to find those imaging marker genes that encode antiatherosclerosis proteins or that can activate antiatherosclerotic prodrugs. Another strategy is to build a bicistronic gene vector, a vector that carries and expresses two genes simultaneously—an imaging marker gene and a therapeutic gene expressed from the same messenger RNA or the same promoter within the same vector. Thus, with the bicistronic "constructs," one should be able to assess the presence of a functional therapeutic gene by directly detecting the functional imaging marker gene that is simultaneously expressed from the same vector.

	IMAGING PHENOTYPIC CONSEQUENCES OF VASCULAR GENE THERAPY

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
Conventional vascular radiologic techniques such as digital subtraction angiography and US, along with advanced MR angiography and CT angiography, still play a primary role in the assessment of the phenotypic success of vascular gene therapy. These imaging methods enable convenient assessment of reperfusion of the vasculature in the tissues and organs distal to the gene-targeted vessels. For example, gene transfer of plasmid DNA encoding VEGF brings about clinical benefits such as rest pain abolition, limb salvage, and ischemic ulcer healing. These benefits are associated with angiographic evidence of new collateral vessels and improved blood flow in the leg as monitored with MR angiography (30).

Functional MR imaging, such as perfusion MR imaging, provides a useful and sensitive imaging method to evaluate circulatory improvement in tissues and organs (114,115). Future applications of MR imaging technology in the genetic management of cardiovascular disease should focus not only on identification of arterial disease at an early stage with high-spatial-resolution MR angiography but also on assessment of the beneficial effects of vascular gene therapy by using functional MR imaging (116).

	CONCLUSION

TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 
More than 4,000 patients have been enrolled in over 400 clinical gene therapy trials worldwide over the past decade (8). These clinical trials include gene therapy for various cardiovascular diseases, including chronic cardiac and limb ischemia syndromes, vasculoproliferative disorder (eg, after angioplasty or in-stent restenosis), venous graft diseases, hyperlipidemia, and hypercholesterolemia (7,15). Advanced research on gene therapy has also focused on other cardiovascular problems, including thrombosis, primary and transplant-related atherosclerosis, hypertension, and myocardial diseases (eg, congestive heart failure) (7,15).

In recent years, rapidly developing research has provided the potential to use different noninvasive, real-time, high-spatial-resolution imaging modalities to monitor, guide, and enhance vascular gene therapy in vivo. Future efforts need to focus on the development of new vascular imaging markers and strategies for imaging marker signal amplification, as well as improvement of in vivo imaging technologies with an emphasis on validation and translation to clinical practice. This requires multidisciplinary collaboration between radiologists, biologists, chemists, and physicists. Current activities in vascular gene therapy by radiologists are far behind those by practitioners in other specialties, specifically our colleagues in cardiology. Modern imaging techniques provide an important opportunity to monitor and direct vascular gene therapy. Radiologists should play a leading role not only in developing and mastering endovasuclar genetic interventions but also in assessing the success of vascular gene therapy and directing the further refinement of vascular gene therapy technology.

	ACKNOWLEDGMENTS

 
The author thanks the entire research project team working on imaging of vascular gene therapy at Johns Hopkins University for their outstanding work through the years and Mary McAllister for her editorial assistance.

	FOOTNOTES

 
² 9_*: Vascular system, location unspecified.

Abbreviations: GFP = green fluorescent protein, RFP = red fluorescent protein, VEGF = vascular endothelial growth factor

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TOP ABSTRACT INTRODUCTION BASIC CONCEPT OF VASCULAR... APPROACHES TO VASCULAR GENE... GENERAL CONCEPT OF IMAGING... MONITORING OF PRIMARY VASCULAR... IMAGING-ENHANCED VASCULAR GENE... IMAGING FOR SITE-SPECIFIC... TRACKING VASCULAR GENE... COMBINING IMAGING MARKERS AND... IMAGING PHENOTYPIC CONSEQUENCES... CONCLUSION REFERENCES

 

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