Molecular Imaging of Gene Expression and Cell Therapy

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Chapter 45 Molecular Imaging of Gene Expression and Cell Therapy

Joseph C. Wu, Sanjiv Sam Gambhir

INTRODUCTION

In 1953, Watson and Crick elucidated the structure of deoxyribonucleic acid (DNA) in a 2-page letter published in the journal Nature.1 Over the next 50 years, advances in genomic science and molecular medicine have resulted in a better understanding of the pathophysiology of many diseases. Recently, another revolution has been taking place. A new set of technologies, as part of a new field termed molecular imaging, is now being developed to noninvasively examine the integrative functions of molecules, cells, and organs in intact whole-body systems.2,3 The field remains in its infancy at present and to a large extent is limited to the laboratory environment. Notable exceptions are in the disciplines of oncology, neurology, and cardiology, which have started to make the transition from basic science to clinical application. Cardiac imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), positron emission tomography (PET), and ultrasound have seen major advances over the past decades. In the clinical setting, these modalities can provide outstanding data regarding organ structure and physiologic function. This chapter serves as an introduction to the field of cardiovascular molecular imaging, with specific emphasis on its utility in evaluating gene and cellular therapy. Because the field may be new to many, we have purposely simplified much of the discussion on technical details to make the context more appealing to a broad range of readers with different backgrounds.

FUNDAMENTALS OF GENE EXPRESSION

The central dogma of molecular biology states that genetic information flows from DNA to ribonucleic acid (RNA) to protein. The human genome has 23 pairs of chromosomes containing approximately 3 billion base pairs of DNA and ~23,000 genes.4,5 Each gene consists of sequences of four different nucleotides: adenine (A), guanine (G), cytosine (C), and thymine (T). The flow of information from DNA to RNA within the cell nucleus is termed transcription. Each messenger RNA (mRNA) is composed of intron and exon sequences. Introns are intervening sequences that are spliced out and removed. Exons are sequences that exit the nucleus to the cytoplasm to serve as templates for protein synthesis in a process called translation. The proteins are formed from a combination of 20 different units called amino acids. Proteins are considered the workhorse of cell machinery, performing activities ranging from gene regulation to cell integrity to receptor signaling. The versatility of molecular biology cloning techniques allows both the DNA and RNA to be manipulated. For example, restriction enzymes can be used to cleave the DNA of interest at specific sites to generate discrete gene fragments.6 They are named after the bacteria from which they were isolated (e.g., Eco RI from Escherichia coli and Hind III from Haemophilus influenza) and serve originally as part of the bacterial immune arsenal to protect itself against invasion by cleaving the DNA of foreign vectors. These techniques have been used to “cut and paste” therapeutic genes and reporter genes, as will be discussed later. For an in-depth discussion on the basics of molecular biology and genomics, please refer to a recent review article.

BACKGROUND OF CARDIOVASCULAR MOLECULAR IMAGING

Traditionally, researchers have monitored cardiac gene transfer by using reporter constructs such as β-galactosidase (β-gal),7 green fluorescent protein (GFP),8 and chloramphenicol-acetyl transferase (CAT).9 Cellular transplant therapies have employed similar techniques as well as newer approaches such as TaqMan reverse transcriptase polymerase chain reaction (RT-PCR),10 Y-chromosome paint probes,11 and antibodies specific to various stem cell types.12 All of these established traditional techniques, however, require invasive biopsies and/or postmortem tissue sampling for analysis. Molecular imaging offers distinct advantages by allowing for noninvasive, quantitative, and repetitive imaging of targeted macromolecules and biological processes in living organisms.2 Although a vast array of molecular imaging techniques are available, they all require two fundamental elements: (1) a molecular probe that can signal confirmation of gene expression by detecting messenger ribonucleic acid (mRNA) transcripts or proteins and (2) a method to monitor these probes or events. Presently, the two most widely used strategies are direct and indirect imaging.

Direct molecular imaging involves direct probe-target interaction. Targets can include receptors, enzymes, or mRNA. For probe-receptor imaging, radiolabeled monoclonal antibodies binding to tumor cell-specific surface antigens have been used for the past 2 decades.13 Recent examples of cardiac application involved imaging αvβ3 integrin receptor14 or vascular endothelial growth factor (VEGF) receptor15 expressed during angiogenesis after myocardial infarction (MI). For probe-enzyme imaging, the most well-known cardiac application is fluorine-18 (18F)-labeled-fluorodeoxyglucose (FDG), used to assess for myocardial tissue viability. After transport across an intact cell membrane, the 18F-labeled glucose analog undergoes phosphorylation by hexokinase and is retained intracellularly in proportion to the rate of cellular glycolysis.16 The radioactive 18F undergoes positron annihilation into two high-energy γ rays (511 keV), which can be detected as coincidence events by PET. For probe-mRNA imaging, radiolabeled antisense oligonucleotide (RASON) probes can be used.17,18 RASON probes are typically 12 to 35 nucleotides long and are complementary to a small segment of the target mRNA. However, the RASON approach is limited at this point due to (1) low number of target mRNA (~1000 copies) per cell compared to proteins (>10,000 copies); (2) limited tracer penetration across the cell membrane; (3) poor intracellular stability; (4) slow washout of unbound oligonucleotide probes; and (5) low target-to-background ratios. On the other hand, direct imaging of DNA (two copies) within the nuclear membrane has proven exceedingly difficult and is not yet feasible. In addition, knowing the activity of gene expression (as reflected in mRNA transcripts or protein levels) rather than the number of DNA copies is more relevant for biological research. The main disadvantage of direct imaging is that it requires synthesizing a customized probe for the product (e.g., receptor, enzyme, or mRNA) of every therapeutic gene of interest, which can be time-consuming and is not generalizable to most applications.

Indirect molecular imaging using reporter genes has only been recently validated. The concept of imaging reporter gene expression is illustrated in Figure 45-1. A reporter gene is first introduced into target tissues by viral or nonviral vectors. Using molecular biology techniques, the promoter or regulatory regions of genes can be cloned into different vectors to drive reporter gene mRNA transcription. Promoter activity can be “constitutive” (always on), “inducible” (turned on or off), or “tissue specific” (expressed only in the heart, liver, or other organs). Translation of mRNA leads to a reporter protein that can interact with the reporter probe. This interaction may be enzyme based or receptor-based. Probe signals can then be detected by various imaging modalities such as an optical charged coupled device (CCD) camera, PET, SPECT, or MRI.

image

Figure 45-1 Four different strategies of imaging reporter gene/reporter probe. A, Enzyme-based bioluminescence imaging. Expression of the firefly luciferase reporter gene leads to the firefly luciferase reporter enzyme, which catalyzes the reporter probe (D-luciferin) that results in a photochemical reaction. This yields low levels of photons that can be detected, collected, and quantified by a charge-coupled device (CCD) camera. B, Enzyme-based PET imaging. Expression of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene leads to the thymidine kinase reporter enzyme (HSV1-TK), which phosphorylates and traps the reporter probe [18F]-FHBG intracellularly. Radioactive decay of 18F isotopes can be detected using PET. C, Receptor-based PET imaging. The [18F]-FESP is a reporter probe that interacts with the dopamine 2 receptor (D2R) to result in probe trapping on or in cells expressing the D2R gene. D, Receptor-based MRI imaging. Overexpression of engineered transferrin receptors (TfR) results in increased cell uptake of the transferrin-monocrystalline iron oxide nanoparticles (Tf-MION). These changes result in a detectable contrast change on MRI.

Clearly, the main advantage of the reporter gene system is its flexibility and multiplexing capacity.19 By altering various components, the reporter gene can provide information about the regulation of DNA by upstream promoters, intracellular protein trafficking, and the efficiency of vector transduction on cells. Likewise, the reporter probe itself does not have to be changed in order to study a new biological process, saving time and resources required for synthesis, testing, and validation of new reagents. However, the main disadvantage of indirect imaging is that it remains a surrogate marker for the physiologic/biochemical process of interest, as opposed to directly measuring receptor density, mRNA copies, or intracellular enzymatic activity, which might be more clinically relevant. It should be noted that in the case of monitoring stem cell fate, this is less of a concern, since the focus is on detecting the presence of a population of cells. Finally, the ideal reporter gene and/or reporter probe should have the following characteristics:

1. The chromosomal integration or episomal expression of reporter gene should not adversely affect the cellular metabolism or physiology.
2. The reporter gene product should not elicit a host immune response.
3. The size of the promoter/enhancer elements and reporter gene should be small enough to fit into a delivery vehicle.
4. Transfection (e.g., plasmid or adeno-associated virus) or transduction (e.g., lentivirus or retrovirus) using the delivery vector should not be cytotoxic to the cells.
5. The reporter probe should be stable in vivo and reach the target site despite natural biological barriers (e.g., blood vessel wall or blood-brain barrier).
6. The reporter probe should only accumulate within cells that express the reporter gene to yield a high signal-to-background ratio.
7. Afterward, the reporter probe should clear rapidly from the circulation to allow repetitive imaging within the same living subject.
8. The reporter probe or its metabolites should not be cytotoxic to the cells.
9. The image signals should correlate well with true levels of reporter gene mRNA and protein in vivo.
10. The reporter gene and reporter probe should be potentially applicable for human imaging in the future.

At present, no single reporter gene and/or reporter probe assay meets all of these criteria. Thus, the optimal choice of which reporter gene and probe assay to use will depend on the particular application, organ system, and imaging modality available at a given institution.

IMAGING TECHNIQUES

Of the numerous molecular imaging modalities available for monitoring genetic and cellular activity, radionuclide-based assays (e.g., PET, SPECT, planar scintigraphy) are the most useful clinically. There are a number of other devices being used to monitor biological processes in animal models of diseases. Considerable efforts have been made towards the development of miniaturized, small-animal imaging systems such as CCD cameras for bioluminescence/fluorescence, ultrasound, SPECT, PET, and MRI. Given the vast array of imaging tools available for biological research, a brief discussion highlighting the strengths and weaknesses of each modality is warranted.

Optical Imaging

Bioluminescence imaging utilizes the photogenic properties of various luciferase genes cloned from different organisms, such as firefly (Photinus pyralis), jellyfish (Aequorea), coral (Renilla), and dinoflagellates (Gonyaulax). In the case of the firefly, the firefly luciferase enzyme (FL) converts its substrate (D-luciferin) to oxyluciferin via an ATP-dependent pathway. This process emits low levels of photons (2 to 3 eV) that can be detected and counted by a cooled CCD camera (e.g., Xenogen IVIS system).20 Unlike bioluminescence, fluorescence imaging does not require injection of a reporter substrate but relies upon an excitation wavelength that produces an emission wavelength for measurement.21 Recent technologic advances (e.g., eXplore Optix system) has allowed the measurement, quantification, and visualization of fluorescence intensity in small living animals using the time domain approach.22 In general, optical-based imaging is a relatively low-cost endeavor (i.e., typically $100 to $200K versus $500K to $1 million for small-animal PET and MRI systems) with the capacity for high throughput (i.e., several mice can be scanned at once). However, the aforementioned techniques suffer from low spatial resolution, inability to monitor multiple physiologic processes, and photon attenuation/scatter within deep tissues.23 Moreover, optical imaging has yet to be extrapolated into clinical usage. Use of novel intravascular catheter devices capable of detecting bioluminescence and/or fluorescence signals from deeper tissues or organs may be possible, but the general practicality of “invasive imaging” remains to be seen.24

Magnetic Resonance Imaging

Unlike optical imaging, MRI has the advantage of a very high spatial resolution (25 to 100 μm) and the ability to measure more than one physiologic parameter at once by using different radiofrequency pulse sequences.25 This makes MR a very attractive option for imaging reporter gene expression. The imaging signal is generated as a result of spin relaxation effects, which can be altered by atoms with high magnetic moments (e.g., gadolinium and iron). One particularly useful MR imaging signal amplification system is based on the cellular internalization of superparamagnetic probes such as monocrystalline iron oxide nanoparticles (MION) and crosslinked iron oxide (CLIO).26 MIONs or CLIOs can be linked to a variety of biomolecules to produce injectable probes for targets such as hematopoietic and neural progenitor cells,27,28 activated thrombotic factor XIII,29 and endothelial cell surface markers such as E-selectin.30 These studies hold promise for in vivo imaging in humans, given the widespread availability of clinical MR scanners, nontoxic and biodegradable properties of intravenous superparamagnetic particles, and the precedent of similar preparations already in clinical use.31 However, persisting residual signals from superparamagnetic particles may hinder the capacity for quantitative and repetitive imaging. MR is also several log of orders less sensitive (10−3 to 10−5 Molar) for detection of reporter probes compared to optical bioluminescence imaging (10−15 to 10−17) or PET (10−11 to 10−12) imaging.2 Therefore, further strategies for robust signal amplification will be necessary before this modality can be of practical use for imaging cardiac gene expression and detecting small numbers of transplanted cells.

Ultrasound

The scope of ultrasound for cardiac molecular imaging has seen increasing applications over the past decade.32–34 Targeted contrast agents have been constructed by linking ligands of interest to liposomes, perfluorocarbon nanoparticles, and encapsulated microbubbles,35 but these agents are relatively large, precluding their extravasation from the vasculature. While ultrasound-based molecular imaging may play an increasing role in endothelial imaging,36 its utility as a molecular cardiac imaging modality to track gene expression or cell therapy remains to be seen. Newer techniques in photoacoustic molecular imaging where the molecular imaging agents can extravasate from blood vessels may hold a potential solution to limitations of conventional ultrasound.37

Computed Tomography

CT is quite limited in its application as a true molecular imaging modality. Development of radiopaque probes that can accumulate in sufficient quantities for meaningful assessment of physiologic processes has proven difficult. Compared to MRI, CT-based images exhibit poor soft-tissue contrast, necessitating iodinated contrast media in addition to any probes that might be used. Presently, CT is best reserved for use as an adjunct anatomic imaging modality that can complement functional information obtained by other molecular imaging techniques, as will be discussed later. In the future, improved probes may also increase the applicability of CT imaging.

Radionuclide Imaging (See Chapter 11)

Radionuclide probes are the first example of molecular probes used in the clinical setting, and this technology represents the evolutionary roots of molecular imaging as it is known today. PET, SPECT, and planar scintigraphy have all been used to detect radionuclide-labeled probes. However, PET exhibits several advantages compared to other modalities. First, PET is more sensitive compared to SPECT and MRI for detection of probe activity, as already discussed. This may allow monitoring of gene delivery by vectors with relatively low transfection efficiencies (e.g., plasmid) or weak promoters (e.g., tissue specific), as well as detection of low numbers of cells (e.g., cardiac stem cell transplants). Second, PET imaging is more quantitative (unlike MRI) and allows for dynamic imaging with tracer kinetic modeling for analysis of rate constants underlying the biochemical processes.38 Third, inasmuch as many PET tracers have a short half-life (e.g., ~110 minutes for 18F), daily repetitive imaging of tracer retention by targeted tissues is possible. Fourth, PET imaging is tomographic (unlike two-dimensional (2D) images from optical imaging), so a relatively precise location of probe signal can be identified within the heart. This is especially apt in the basic research environment, because current generations of small-animal PET scanners have a resolution of 13 to 23 mm3 compared to around 63 mm3 for clinical PET scanners.39 Finally, studies performed in these small-animal PET scanners can potentially be scaled up to human patients using clinical PET scanners with relative ease.3

IMAGING CARDIAC GENE THERAPY

Gene transfer has been heralded as the most promising therapy of molecular medicine in the 21st century. It is usually defined as the transfer and expression of DNA to somatic cells of an individual, with a resulting therapeutic effect (see Fundamentals of Gene Expression section). In cardiovascular diseases, gene therapy offers an exciting new approach to express the therapeutic factors locally in the myocardium.40 In general, the successful application of gene therapy requires three essential elements: (1) a vector for gene delivery, (2) delivery of the vector to the target tissue, and (3) a therapeutic gene to be expressed in a particular patient population.

An ideal vector should enable efficient gene delivery to the target tissue, have minimal local or systemic toxicity, deliver enough concentration and duration to induce a therapeutic effect, and cause no germline transmission to the offspring. No single vector has all of these attributes, so the type of vector chosen will need to be tailored to the specific clinical applications. Vectors can be divided into viral vectors and nonviral vectors. Common viral vectors include adenovirus, adeno-associated virus, gutless adenovirus, and lentivirus. Nonviral vectors include plasmids, minicircles, naked DNA, and liposomes. The technique of vector delivery to the heart also depends on the intended target area, but in general can be categorized as: (1) direct epicardial injection, (2) endocardial injection, (3) intracoronary infusion, (4) retrograde coronary sinus infusion, and (5) pericardial injection.

Likewise, the choice of a therapeutic gene to be expressed is often driven by the intended application. In animal studies, successful gene therapy has been demonstrated for:

1. Treatment of coronary artery disease (CAD) using angiogenic factors such as VEGF,41 fibroblast growth factor (FGF),42 and hypoxia-inducible factor-1α (HIF-1α)43
2. Reduction of restenosis after angioplasty by inhibiting smooth muscle cell proliferation by suicide gene therapy using thymidine kinase44
3. Improvement of congestive heart failure by gene transfer of calcium ATPase pump (SERCA2a)45
4. Inhibition of atherosclerosis by overexpression of HDL receptor46
5. Reduction of hypoxia-induced apoptosis of cardiomyocytes47

These encouraging results have led to the initiation of several clinical trials, beginning in the 1990s. Of the 509 ongoing gene therapy trials in the United States, 46 are related to cardiovascular diseases. The majority of these are aimed at testing the safety and efficacy of therapeutic angiogenesis and, to a lesser extent, examining restenosis. In the late 1990s, several phase-1 open-labeled trials involving small numbers of patients with myocardial ischemia and peripheral vascular disease uniformly showed positive results.48–50 However, recent phase-2 randomized, double-blind, placebo-controlled trials have yielded conflicting, if not disappointing, results. The Vascular Endothelial Growth Factor in Ischemia for Vascular Angiogenesis (VIVA),51 FGF Initiating Revascularization Trial (FIRST),52 Adenovirus Fibroblast Growth Factor Angiogenic Gene Therapy (AGENT),53,54 and Kuopio Angiogenesis Trial (KAT)55 tested gene therapy using either VEGF or FGF. Unfortunately, these trials failed to show any consistent improvement in various parameters such as symptomatic improvement, ejection fraction, wall motion scores, myocardial perfusion, and restenosis rate.

Nonetheless, important lessons can be learned from these trials. They showed that angiogenesis is a complex process regulated by the interaction of various growth factors and may be difficult to stimulate using a single protein or gene injection. The ideal injection method, delivery vector, and patient population remain to be determined. The pharmacokinetics and pharmacodynamics of therapeutic gene expression will need to be defined first before gene therapy can proceed further to widespread clinical usage, similar to the research and development of experimental drugs.56 Finally, since there is no available method of assessing gene expression in vivo, the investigators are unable to determine whether the lack of symptomatic improvement is due to poor injection technique, insufficient gene expression, host inflammatory response, or an inappropriate therapeutic gene.

Imaging of cardiac transgene expression has been established in several proof-of-principle studies involving injection of various reporter genes into the myocardium and following the kinetics of transgene expression over time, using optical bioluminescence, microPET, and clinical PET imaging.57–61 The feasibility of linking a PET reporter gene to a therapeutic gene was also demonstrated in two separate studies.62,63 In this case, an adenovirus containing two constitutive cytomegalovirus promoters driving a VEGF121 therapeutic gene and an HSV1-sr39tk PET reporter gene separated by polyadenine sequences was constructed (Ad-CMV-VEGF121-CMV-HSV1-sr39tk). Wu et al. injected the construct into the myocardium of a rat in a myocardial infarction model.63 Reporter gene expression (which indirectly reflects the VEGF121 therapeutic gene expression) persisted for only about 2 weeks, owing to host immune response against the adenovirus. At 2 months, there were no significant improvements in myocardial contractility, perfusion, or metabolism, as measured by echocardiography, nitrogen-13-labeled ammonia (13N-NH3), and FDG imaging, between study and control groups (Fig. 45-2). Thus, this study highlights the importance of monitoring the pharmacokinetics of gene expression. It also demonstrates the proof of principle that any other cardiac therapeutic genes of interest (e.g., HIF-1α, SERCA2a, heat shock protein, or endothelial nitric oxide synthase) can likewise be coupled to a PET reporter gene for subsequent noninvasive monitoring.

image image

Figure 45-2 PET imaging of cardiac angiogenic gene therapy. A, Schematic of Ad-CMV-VEGF121-CMV-HSV1-sr39tk–mediated gene expression. Two separate gene cassettes with CMV promoters driving the expression of a VEGF121 therapeutic gene and an HSV1-sr39tk reporter gene separated by polyA tails. The translated product of VEGF121 is soluble and excreted extracellularly, whereas the translated product of HSV1-sr39tk (HSV1-sr39Tk) traps 18F-FHBG intracellularly by phosphorylation. B, Noninvasive imaging of the kinetics of cardiac transgene expression. Gene expression peaked at day 1 and rapidly decreased thereafter. A second injection (arrow) of Ad-CMV-VEGF121-CMV-HSV-sr39tk at day 60 yielded no detectable signal on day 62 and day 64. Error bars represent mean ± SEM. C, A representative rat scanned longitudinally with transaxial 18F-FHBG PET images shown at similar slice levels of the chest cavity. The gray scale is normalized to the individual peak activity of each image. In this rat, myocardial [18F]-FHBG accumulation was visualized at the anterolateral wall (arrow) from day 1 to day 14 but not days 17, 62, or 64. D, In vivo gene, perfusion, and metabolism imaging with PET. At day 2, representative images showing normal perfusion ([13N]-NH3) and metabolism ([18F]-FDG) in a sham rat, anterolateral infarction in a control rat (Ad-null), and anterolateral infarction in a VEGF-treated study rat (Ad-CMV-VEGF121-CMV-HSV1-sr39tk) in short, vertical, and horizontal axis (gray scale). The color scale is expressed as % ID/g for [18F]-FHBG uptake. As expected, both the sham and control animals had background 18F-FHBG signal only (blue color) that outlined the shape of the chest cavity. In contrast, the study rat showed robust HSV1-sr39tk reporter gene activity near the site of injection.

(From Wu JC, Chen IY, Wang Y, et al: Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium, Circulation 110:685–691, 2004. Reproduced with permission.)

The feasibility of transferring imaging results from a small-animal PET scanner to a clinical PET scanner (ECAT EXACT scanner, Siemens/CTI) has been demonstrated in porcine models as well. Bengel et al. showed that myocardial tissues infected with adenovirus expressing HSV1-tk had significantly higher iodine-124-labeled 2′-fluoro-2′-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil (124I-FIAU) retention during the first 30 minutes following injection.60 The FIAU uptake correlated with ex vivo images, autoradiography, and immunohistochemistry for reporter gene product after euthanasia. However, the signal-to-background ratio at the site of HSV1-tk injection was only around 1.25 during the first 30 minutes following delivery. Afterwards, there was significant washout at 45 to 120 minutes post injection, and the 124I-FIAU retention became similar to control myocardial regions. Recently, Rodriquez-Porcel et al. also demonstrated the feasibility of imaging HSV1-tk reporter gene expression with fluorine-18-labeled fluoro-3-hydroxymethyl butylguanine (18F-FHBG) reporter probe in a porcine model. Following endomyocardial injection using a Biocardia catheter, myocardial gene expression activity was quantified by clinical PET-CT system (Fig. 45-3). The best myocardial signal-to-background (left ventricular) ratio was obtained 3 hours post-injection (180 minutes, 4.63 ± 1.4, versus 90 minutes, 1.78 ± 0.6; P < 0.05). Autoradiography and microPET confirmed the increased 18F-FHBG uptake in the anteroseptum. Overall, these studies suggest that the combination of HSV1-tk/FHBG may be superior to HSV1-tk/FIAU for imaging cardiac transgene expression.64

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Figure 45-3 Imaging of HSV-1tk/[18F]FHBG in preclinical large-animal model. Percutaneous delivery system consisting of (A) steerable guiding catheter and (B) helical needle infusion catheter. A steerable catheter provides maximum flexibility, allowing catheter positioning in virtually any area of the left ventricular cavity. The infusion catheter is used first to confirm intramyocardial positioning of the catheter and then for the delivery of therapeutic material. C, The infusion catheter is “screwed’ inside the myocardium. D, Representative PET/CT image 48 hours after percutaneous gene delivery. A, anterior; B, bladder; CL, contralateral; I, intestines; K, kidney; L, left; Li, liver; LV, left ventricular; P, posterior; R, right.

(From Rodriguez-Porcel M, Brinton TJ, Chen IY, et al: Reporter gene imaging following percutaneous delivery in swine moving toward clinical applications, J Am Coll Cardiol 51:595–597, 2008. Reproduced with permission.)

In the future, it will also be useful to have the following arsenals for cardiac gene therapy: (1) non-immunogenic vectors such as non-viral minicircle plasmids that can prolong transgene expression in the myocardium,65 (2) cardiac tissue-specific promoter (e.g., myosin light chain kinase or troponin) that can diminish unwanted extracardiac activity,66 (3) development of novel gene therapy approaches such as short hairpin RNA interference,67 and (4) multimodality molecular imaging approaches that can monitor the location, magnitude, and duration of transgene expressions, as well as their downstream functional effects.56

IMAGING CARDIAC CELL DELIVERY

In recent years, research in stem cell therapy has rivaled or even surpassed gene therapy as a promising treatment for ischemic heart disease. In fact, stem cell therapy now occupies the lion’s share of scientific presentations, media attention, and the public imagination. By most accounts, this field “started” in 2001 when Orlic et al. published a seminal (albeit still controversial to date) study showing that a particular type of bone marrow cells (lin−/c-kit+) could form de novo myocardium occupying 68% of the infarcted left ventricle just 9 days after transplantation in a mouse model.68 At present, there are many studies reporting the efficacy of fetal and neonatal cardiomyocytes,69 skeletal myoblasts,70,71 bone marrow cells (BMCs),68 and embryonic stem cell derivatives72 for myocardial repair. As with any type of therapy, each cell type also has its own merits and pitfalls.73

Fetal and neonatal cardiomyocytes have been regarded as promising candidates for cell replacement therapy,69 but their restricted availability and ethical dilemma render widespread clinical application impractical. Autologous skeletal myoblasts were the first cell type to be used clinically for cell-based cardiac repair.70 Skeletal myoblasts are attractive candidates because they can be cultured and expanded ex vivo from muscle biopsies, and they survive well after transplantation because of their strong resistance to ischemia. Skeletal myoblast transplantation has been shown to provide functional benefit in animal models of infarction, but a large placebo-controlled, randomized trial in humans did not demonstrate sustained efficacy as defined by the primary end point of left ventricular ejection fraction (LVEF).74 Furthermore, the increased number of early postoperative arrhythmic events after skeletal myoblast transplantation raises serious concerns and warrants further investigation. By contrast, transplantation of BMCs has been shown to improve heart function in animal studies, and no serious complications have been reported in clinical trials to date. However, results from three large clinical trials published in 2006 in the New England Journal of Medicine showed inconsistent benefits that at best led to short-term but no long-term improvement.75–77 In the accompanying editorial, the author urges the scientific community to “guard against both premature declarations of victory and premature abandonment of a promising therapeutic strategy” and that “this strategy is likely to depend on continued and effective coordination of rigorous basic and clinical investigations.”78 More recently, isolation of resident cardiac stem cells from human biopsy samples has been demonstrated.79 Whether these cells can promote cardiac regeneration and improve heart function in humans following transplantation will need to be addressed in future clinical trials.

One of the main limitations of cardiac stem cell therapy is the lack of available methods to assess stem cell survival. In clinical studies, changes in myocardial perfusion, viability, and perfusion are assessed by echocardiography, nuclear SPECT and PET imaging, or MR imaging. These parameters measure the therapeutic effects but do not actually detect the presence or absence of transplanted stem cells. This is an important distinction because most patients also undergo concurrent coronary artery bypass grafting (CABG) or percutaneous coronary intervention (PCI), and it is not clear whether the improvement is due to these procedures or to the transplanted stem cells. Likewise, in animal studies, analysis of stem cell survival is based on postmortem histology, which is invasive and precludes longitudinal monitoring. Thus, the ability to study stem cells in the context of the intact whole-body system rather than by indirect (clinical trials) or invasive (animal studies) means will give better insight into the underlying biology and physiology of stem cells. Several investigators have started addressing this issue via different approaches, including radionuclide labeling, ferromagnetic labeling, and reporter gene labeling (Fig. 45-4).73

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Figure 45-4 Overview of techniques used for noninvasive imaging of stem cell fate. The four different techniques include magnetic particle labeling, radionuclide labeling, quantum dot labeling, and reporter gene labeling. IFP, iron fluorescent particles; SPIO, superparamagnetic iron oxide.

(From Zhang SJ, Wu JC: Comparison of imaging techniques for tracking cardiac stem cell therapy, J Nucl Med 48:1916–1919, 2007. Reproduced with permission.)

Radionuclide Labeling

This technique requires the transplanted cells to be directly labeled with a radioisotope prior to transplantation. The advantages are high sensitivity and immediate translation to clinical practice because indium-111 (111In)-oxine and 18F-FDG are approved by the U.S. Food and Drug Administration (FDA) as clinical radiotracers. The technique has demonstrated utility for tracking cell homing in, on infarcted myocardium and distribution following injection. Aicher et al. demonstrated that 111In-oxine-labeled endothelial and hematopoietic progenitor cells accumulated in infarcted rat myocardium after intraventricular injection.80 However, by 96 hours postinjection, only about 5% of the injected dose of radioactivity was found in the heart. This highlights the sensitivity of the method, because although only a few cells persisted in the heart, they were nevertheless detectable. An inherent limitation of radionuclide labeling is the short half-life of the radioactive probes, which precludes long-term imaging. For radiotracers with a shorter half-life (e.g., 18F-FDG is 110 minutes), one can follow those cells at most, out to 1 day. Even for radiotracers that have longer half-lives (e.g., copper-64 [64Cu], 12.7 hrs; [111In], 67.3 hours; thallium-201 [201Tl], 74 hours; [124I], 96 hours), the longest time period of imaging would still be limited to within the first 1 to 2 weeks. Furthermore, the radiotracer itself has other concerns, specifically potential adverse effects of the tracer on stem cell viability and differentiation and the leakage of the tracer from the labeled cells, which can provide a false-positive signal.81

Nanoparticle Labeling

Semiconductor quantum dots (QDs) are a new class of fluorescent probes that have been used in noninvasive imaging in recent years.82,83 This technique makes use of fluorescent semiconductor nanocrystals to detect membrane molecules of interest. The excitation wavelengths of the QDs can be manipulated, ranging from ultraviolet to near-infrared ranges. Depending on the size and composition of these probes, they can be designed to emit different wavelengths of light. The photostability of QDs, as seen in their resistance to photobleaching and long-lasting fluorescence, makes them appealing for tracking stem cells in vivo.84 However, the effects of QDs on stem cell proliferation and differentiation remain unclear; mixed results have been reported using different origins of stem cells or experimental protocols.82,85 Finally, several other obstacles, including the tendency for aggregation of QDs in the cytosol, difficulty in delivery of QDs into cells, and nonspecific binding to multiple molecules,86 must be overcome before QDs can realize their full potential in clinical imaging. More recently, Raman nanoparticles have also started to be imaged in small living subjects and may provide as much as 100-fold greater sensitivity than QD-based approaches.87,88

Iron Particle Labeling

Compared to radionuclide imaging, the main advantage of MRI is its capacity for high anatomic resolution. In general, MRI techniques can be divided into two classes based on the contrast agent being used. Lanthanide gadolinium (Gd3+) is applied for T1-weighted contrast enhanced images, and superparamagnetic iron oxide (SPIO) particles are applied for T2-weighted images. Gadolinium-enhanced images have been used in previous studies to track human neural and mesenchymal stem cells for up to 6 weeks.89 However, SPIO particles are favored and more widely used for stem cell imaging because a lower concentration (5–10 µm/L) is required. The immediate translation of SPIO to clinical studies is feasible because certain superparamagnetic formulations have already been approved by the FDA and found to be biocompatible, safe, and nontoxic. Recent studies have demonstrated the utility of MRI for long-term tracking of bone-marrow-derived mesenchymal stem cells (MSCs). Amado et al. labeled porcine MSCs with iron oxide, induced a myocardial infarct (MI) by balloon occlusion of the left anterior descending (LAD) artery, and then injected MSCs intramyocardially 3 days after MI.90 The iron oxide–labeled cells created hypoenhanced regions. Interestingly, relative to the intensity of hypoenhancement at 2 days posttransplantation, approximately 42% of the signal still remained at 8 weeks. Similarly, Kraitchmen et al. magnetically labeled porcine MSCs with iron oxide, injected the cells into adult swine following MI, and demonstrated that the cells could be tracked for up to 3 weeks using MRI.91 MRI showed that the cell survival trend correlated with proximity to the infarct; specifically, cells injected into the infarct zone had more robust survival than those injected into healthy myocardium. In a related study, Kraitchmen et al. compared the sensitivity of SPECT to MRI to detect MSCs injected intravenously following MI in canines (Fig. 45-5).92 MSCs were co-labeled with [111In]-oxine and iron oxide, thus enabling SPECT and MRI tracking, respectively. SPECT demonstrated MSCs homing and engrafting to the heart for up to 7 days, whereas MRI was insufficiently sensitive to detect the cells in the heart or any other organ. In summary, MRI currently lacks the sensitivity of radionuclide-based labeling (SPECT/PET), which limits its ability to detect small numbers of cells in the heart (>105 cells are needed to be detectable).92 In addition, the ferromagnetic probe may become diluted with cell division, making it difficult to quantify proliferating cells. Last, the detected signal does not necessarily indicate whether the cells are viable. This is because iron particles can remain internalized in nonviable cells, and the iron particle may be engulfed by tissue macrophages, both events creating a false-positive signal.93,94

image

Figure 45-5 Radionuclide imaging of mesenchymal stem cells following intravenous injection. Sagittal (left) and coronal (right) views of fused SPECT/CT images on days 1 (A), 2 (B), and 7 (C) in an animal that demonstrated focal uptake in the anterior midventricular region of the heart. At the last imaging time point on days 5 to 8 (D), an anterior apical region of MSC uptake (arrow) is shown in three representative animals in the coronal view. This more anterior apical distribution was present independent of whether an early focal hot spot was observed (yellow arrowhead).

(From Kraitchman DL, Tatsumi M, Gilson WD, et al: Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction, Circulation 112:1451–1461, 2005. Reproduced with permission.)

Reporter Gene Labeling

In contrast to physical labeling (radionuclide and iron particles), reporter gene labeling requires genetic modification of the stem cells. In the first proof-of-principle study using reporter genes to track cardiac cell survival, Wu et al. transfected rat H9c2 embryonic cardiomyoblasts with adenovirus carrying either firefly luciferase or HSV1-sr39tk reporter gene before injection into the rat myocardium.95 Cell survival was monitored noninvasively by optical bioluminescence or small-animal PET imaging. Cell signal activity was quantified in units of photons per second per square centimeter per steradian (photons/sec/cm2/sr) or percentage injected dose of 18F-FHBG per gram tissue (%ID/g), respectively. In both cases, drastic reductions in signal activity were seen within the first 1 to 4 days due to acute donor cell death from inflammation, ischemia, and apoptosis. Interestingly, this pattern of cell death was consistent with other reports using traditional ex vivo assays such as TUNEL apoptosis,96 classical histology,97 and TaqMan RT-PCR.10 However, all of these ex vivo techniques required large numbers of animals to be sacrificed at different time points.

Given that several types of reporter genes and reporter probes are now available, it should be possible in the future to perform multimodality imaging of stem cell transplantation. For example, the feasibility of imaging murine embryonic stem cells transplanted into the rodent myocardium has been demonstrated (Fig. 45-6).98 These cells were genetically manipulated to express a triple-fusion reporter that consists of red fluorescence protein (RFP) for fluorescence activated cell sorting (FACS) analysis and single cell fluorescence microscopy, firefly luciferase (Fluc) for high throughput bioluminescence imaging, and herpes simplex virus thymidine kinase (HSVtk) for small-animal PET imaging. Imaging analysis revealed significant intracardiac and extracardiac teratoma formation with transplantation of undifferentiated embryonic stem cells. Thus, future efforts will need to involve differentiated derivatives such as embryonic stem cell–derived endothelial cells99 or cardiomyocytes100 for myocardial repair while avoiding the tumorigenicity issues.101 Follow-up studies have also been demonstrated in a large-animal model using clinical PET/CT imaging of porcine MSCs stably transduced with Renilla luciferase, red fluorescent protein, and herpes simplex virus truncated thymidine kinase (RL-RFP-HSVttk) (Fig. 45-7).102 In addition, several studies have used transgenic mice that constitutively express reporter genes to understand the spatiotemporal kinetics of bone marrow stem cell homing,103 the optimal adult stem cell type for improving cardiac function,104 the efficacy of different biomatrices capable of improving cell survival,105 the fate of resident stem cells isolated from the heart,106 and the effect of acute versus chronic infarction on stem cell viability.107 These multi-reporter gene approaches may be helpful for evaluating other issues relevant to stem cell biology, such as imaging stem cell survival, proliferation, and differentiation using tissue-specific promoters.

image

Figure 45-6 Reporter gene imaging of embryonic stem cells expressing triple-fusion reporter gene. A, To assess longitudinal cell survival, animals were imaged for 4 weeks. A representative study animal injected with ES-TF cells showed significant bioluminescence (top) and PET (bottom) signals at day 4, week 1, week 2, week 3, and week 4. In contrast, control animals had background activities only. B, Quantification of imaging signals showed a drastic increase of fluc and ttk activities from week 2 to week 4. Extracardiac signals were observed during subsequent weeks. C, Quantification of cell signals showed a robust in vivo correlation between bioluminescence and PET imaging (r2 = 0.92). BLI indicates bioluminescence. D, After cell transplant, animals underwent [18F]-FHBG reporter probe imaging (top row) for detection of cell survival and [18F]-FDG imaging (middle row) for assessment of myocardial metabolic activity. Note background bone uptake of free fluoride is seen in both images. Fusion of the two images (bottom row) shows the exact location of transplanted embryonic stem cells carrying the triple-fusion reporter gene (arrow) at the anterolateral wall. The small-animal PET imaging provides horizontal, coronal, and sagittal views. Similar studies should be feasible using human clinical PET scanners in the future.

(From Cao F, Lin S, Xie X, et al: In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation 113:1005–1014, 2006. Reproduced with permission.)

image

Figure 45-7 Reporter gene of stem cell therapy in a large-animal preclinical model. A, Endocardial mapping of a pig heart 16 days after myocardial infarction. Voltage map with sites (black points) of NOGA-guided intramyocardial injections of LV-RL-RFP-tTK-MSC (white arrows at border zone of infarction) and nontransfected mesenchymal stem cells (MSCs) (yellow arrow at noninfarcted posterior wall). Normal viability is represented by blue and pink colors. Yellow and green color represents decreased viability in the mid-distal anterior wall, and red, nonviability at the heart apex. B, [13N]-NH3 PET with transmission scan of the pig heart (supine position) 16 days after acute myocardial infarction, indicating perfusion defect in the anterior wall and apex. C, [18F]-FHBG tracer uptake in the two injected points, representing the location of the LVRL-RFP-tTK-MSC 8 hours after cell delivery into the myocardium (PET transmission scan, pig in supine position). No activity in the posterior wall, where the nontransfected MSCs were injected. D, Fusion image of MRI (grayscale) and [18F]-FHBG-PET (hot scale) indicating tracer accumulation in the sites only where LV-RL-RFP-tTK-MSCs were intramyocardially injected. E, [18F]-FHBG-PET-CT hybrid image for localization of the injected cells in the anterior wall.

(From Gyongyosi M, Blanco J, Marian T, et al: Serial non-invasive in vivo positron emission tomographic (PET) tracking of percutaneously intramyocardially injected autologous porcine mesenchymal stem cells modified for transgene reporter gene expression, Circ Cardiovasc Imaging 1:94–103, 2008. Reproduced with permission.)

Clinical Imaging

The first steps toward using molecular imaging technology to track cardiac cell therapy in humans have already been undertaken. In a recent study by Hofmann et al., PET imaging was used to track [18F]-FDG-labeled autologous bone marrow cells for treatment of patients following acute MI (Fig. 45-8).108 Unselected bone marrow cells were radiolabeled with 100 MBq [18F]-FDG and infused into the infarct-related coronary artery (3 patients), or injected into the antecubital vein (3 patients). In an additional group of 3 patients, a CD34+-enriched population of bone marrow cells was delivered by intracoronary infusion. In all groups, cells were administered 5 to 10 days following coronary stenting, and PET imaging was carried out 50 to 75 minutes after the procedure. PET successfully detected [18F] signals in all groups, with higher intramyocardial signal in the intracoronary versus intravenous delivery groups. Of the two groups receiving intracoronary infusion, the CD34+-enriched population had a higher myocardial signal than unselected BMSCs (1.3% to 2.6% versus 14% to 39%, P < 0.005), suggesting enhanced homing to the injured myocardial milieu associated with CD34+ stem cells (see Fig. 45-8). Although conducted in a small number of patients, this study demonstrates nicely a potential means to track stem cell therapy on a short-term basis. The fact that differing myocardial signal between groups was observed is encouraging insofar as it helps validate the sensitivity of clinical PET scanning for tracking cellular delivery and homing. As mentioned earlier, the major limitation of this technique is its inability to track long-term survival kinetics and cellular trafficking following therapy, given the half-life of 18F-FDG is only around 110 minutes. Furthermore, radiolabeled cell signals do not provide information regarding cell proliferation, because the radiotracers cannot be “passed on” from mother to daughter cells. It would be most useful, for example, to observe changes in myocardial stem cell populations over weeks and correlate their late-phase survival or proliferation with functional improvement. In the future, genetically engineered constructs integrated into the transplanted stem cells may allow for such measurements, using a variety of molecular imaging techniques discussed here. Very recently, the feasibility of imaging CD8+ T cells genetically engineered to express the HSV1-tk and imaged with 18F-FHBG PET has been performed, and this sets the stage for human imaging of cell populations.109 However, significant challenges remain to be overcome. From the imaging standpoint, the main obstacles are understanding the lowest number of detectable stem cells in the heart and the use of other reporter genes (e.g., human dopamine 2 receptor or human sodium iodide symporter or humanized HSVtk) that may be less immunogenic compared to the viral HSV1-tk.61,110,111

image

Figure 45-8 Monitoring of myocardial biodistribution of 18F-FDG-labeled BMC in humans. Left posterior oblique (A) and left anterior oblique (B) views of the chest and upper abdomen of a patient, 65 minutes after transfer of 18F-FDG-labeled, unselected bone marrow cells into the left circumflex coronary artery. BMC homing is detectable in the lateral wall of the heart (infarct center and border zone), liver, and spleen. Left posterior oblique (C) and left anterior oblique (D) views of the chest and upper abdomen of another patient, 70 minutes after transfer of 18F-FDG-labeled, CD34-enriched bone marrow cells into the left anterior descending coronary artery. Homing of CD34+-enriched cells is detectable in the anteroseptal wall of the heart, liver, and spleen. CD34+ cell homing is most prominent in the infarct border zone (arrowheads) but not in the infarct center (asterisk).

(From Hofmann M, Wollert KC, Meyer GP, et al: Monitoring of bone marrow cell homing into the infarcted human myocardium, Circulation 111:2198–2202, 2005. Reproduced with permission.)

CONCLUSION

In summary, various approaches for monitoring cardiac gene and cell therapy have been discussed. Molecular imaging has emerged as a valuable tool for monitoring gene delivery and, more recently, following cellular therapy. Although the field is in its infancy, molecular imaging in humans is likely to grow rapidly as “molecular medicine” tailored to individual patients becomes a clinical reality in the future. Without a doubt, successful therapies will be predicated on a sound understanding of the pharmacokinetics, functional, and biological aspects of gene therapy and cellular delivery. Cardiac molecular imaging is an important tool that can help achieve these goals. The next 5 years should witness great excitement and progress as it has been for the past 5 years.

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