Research Methods

Picture This: Imaging Gene Expression in the Lungs

By Daniel P. Schuster, M.D.

Washington University School of Medicine

Introduction

Picture this: the pre-clinical work is over; the regulatory work is done. Now its time to administer the therapeutic gene to humans to see if it can truly reverse or prevent the progression of pulmonary disease. But wait -- if gene therapy is to be implemented for diseases affecting the lungs, it will be critical first to know that the chosen route of vector administration has effectively transduced lung tissue, that the levels of transgene expression correlate with physiologic effects, and that the duration of gene expression is sufficient to provide the desired physiologic result.

Many issues still bedevil the field of gene therapy, among them the problems just described. However, recent breakthroughs in imaging present the promising possibility of being able to monitor the onset, magnitude and duration of gene expression via a group of emerging noninvasive methods collectively referred to as “molecular imaging.” Molecular imaging, as distinct from structural (anatomic) or functional imaging, aims to characterize noninvasively and quantify cellular and subcellular events in intact organisms. The ability to monitor the expression of transgenes is an important example of molecular imaging. Here we highlight some of these new methods and the potential to apply them to study lung disease. In-depth, general reviews of molecular imaging are available elsewhere (1-8).

Techniques

So far three general approaches have emerged to image transgene expression: optical techniques, magnetic resonance imaging, and radionuclide-based methods. Ultimately the goal of each is to determine the expression level of the target or therapeutic transgene by quantifying a noninvasive imaging signal emitted as the result of a probe that accumulates in tissue because of the activity or affinity of a reporter gene product. If properly calibrated, such measurements should then provide reliable information about the location, magnitude, and timing of transgene expression in multiple tissues, with the additional advantage that the measurements can be made in the same subject at multiple times without having to resort to invasive tissue biopsies.

Genes for both enzymes and membrane receptors have been used as imaging reporter genes. Signal amplification is an important advantage of enzyme-based systems; one enzyme can catalyze many molecules of substrate, each of which contributes to the imaging signal. A disadvantage is that the substrate must penetrate into the cell to gain access to the enzyme, potentially limiting substrate accumulation. While membrane receptors don’t have this problem, signal amplification is reduced or nonexistent because ligand-receptor interactions are usually one-to-one. Receptor-based strategies, therefore, may be less sensitive than enzyme-based reporter methods. The value of any one approach over another is an area of active investigation (4).

Optical Imaging

With optical methods, the products of reporter genes are used to generate light by specific biochemical reactions. With bioluminescence techniques, cells expressing the reporter gene product (e.g., luciferase) oxidize a substrate (e.g., D-luciferin), causing it to emit light. With fluorescence imaging, cells are transduced with a gene (e.g., green fluorescent protein) that emits light upon excitation with an external light source at a specific wavelength. Since mammalian tissues do not emit significant levels of intrinsic light, bioluminescence can be used to generate images with virtually no background noise. Background noise is greater for fluorescent imaging because of autofluorescence (light emission even in tissues not expressing the reporter gene product).

The sensitivity of detecting light emitted from within an organism depends on many factors, including the level of reporter gene expression used to generate the signal, the distance light must travel through tissue from source to detector and the sensitivity of the detection system. Tissue attenuation results in a significant loss of imaging signal, limiting application at present to small animals (e.g., mice). Images are also surface-weighted (i.e., light sources closer to the surface of the animal appear brighter than sources deeper within the animal) and they are planar (instead of tomographic or three-dimensional). Thus, optical imaging generally lacks accurate depth information. However, the low cost of optical imaging, ease of use and ability to scan many animals in a short period of time makes this a highly attractive platform to screen for transgene expression.

Magnetic resonance imaging (MRI)

Magnetic resonance images are generated when nuclides (e.g., ¹H, ³He) within the body are subjected to a radiofrequency (rf) pulse applied at a “resonant” frequency to a subject positioned within a magnetic field. In so doing, the nuclides absorb energy and generate a detectable signal. For the purposes of monitoring transgene expression with MRI, imaging contrast is provided by the tissue accumulation of agents, such as chelated gadolinium or superparamagnetic nanoparticles, that alter MR signal intensity in the presence of the reporter gene product.

MRI strategies have two major advantages over other molecular imaging techniques: exquisite spatial resolution (on the order of 10-50 um) and image production without injuring the tissue. However, MRI-based methods are several orders of magnitude less sensitive than either radionuclide- or optical-based techniques. Nevertheless, various amplification strategies have been successfully employed that permit monitoring of transgene expression (7).

Imaging the lung parenchyma presents some special difficulties for MRI, including the lungs’ relatively low tissue density, the presence of magnetic field inhomogeneities resulting from the many air-tissue interfaces within the lungs, and respiratory and cardiac motion (reducing the spatial resolution advantage of MRI).

Radionuclide-based methods

Gamma camera scintigraphy, single-photon emission computed tomography (SPECT) and positron emission tomography (PET) have all been used as platforms for transgene expression imaging. They differ in their availability, cost, need for specialized infrastructure and technical expertise and in their ability to accurately quantify tissue radioactivity noninvasively. PET imaging, while the most costly, provides the greatest opportunity for quantitative accuracy.

Images are generated with PET by the accumulation of positron-emitting radiopharmaceuticals, administered in trace amounts, in tissues expressing the transgene. Positrons (positively charged electrons) travel a short distance in space, then interact with an ambient electron leading to the annihilation of both particles. Two high-energy photons result from this matter-antimatter interaction, which can be detected by radiation detectors placed around the subject. PET imaging is highly sensitive (level of detection approaches 10^-11 M of tracer) and unlike optical imaging, is isotropic (i.e., can quantify gene expression accurately regardless of tissue depth). Although spatial resolution is at least an order of magnitude worse that MRI, PET imaging is the most likely platform to be used for clinical applications initially.

Application to the Lungs

PET and optical imaging have been used to specifically monitor transgene expression in the lungs of experimental animals (9-11). An example of a PET image of rat lungs expressing a mutant form of the imaging reporter gene HSV-1 thymidine kinase is shown in Figure1. Such studies demonstrate that these methods are extraordinarily sensitive, exceeding even the sensitivity associated with tissue-based methods. They also show that these methods can be used to study the relative efficiencies of different vector delivery systems and the intrapulmonary distribution of gene transfer. However, studies showing the relationships among imaging reporter gene expression, target or therapeutic gene expression, and physiologic effect in the lungs have yet to be reported.

Likewise only the expression of exogenous reporters in the lungs has been imaged so far. However, the same principles apply to endogenously expressed transgenes (i.e., transgenic animals). Use of imaging for this purpose will be limited only by the imagination of the investigator.


References:

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8. Schuster D, Piwnica-Worms D, Kovacs A, Garbow J. Recent advances for imaging the lungs of intact small animals. Am J Respir Cell Mol Biol 2003 in press.
9. Richard JC, Factor P, Welch LC, Schuster DP. Imaging the spatial distribution of transgene expression in the lungs with positron emission tomography. Gene Ther 2003;In press.
10. Richard JC, Zhou Z, Ponde DE, et al. Imaging pulmonary gene expression with positron emission tomography. Am J Respir Crit Care Med 2003; 167:1257-1263.
11. Iyer M, Berenji M, Templeton NS, Gambhir SS. Noninvasive Imaging of Cationic Lipid-Mediated Delivery of Optical and PET Reporter Genes in Living Mice. Mol Ther 2002; 6(4):555-562.

Figure 1.
(A.) Coronal view of a PET image obtained 3 days after intratracheal administration of an adenovector carrying a mutant form of the HSV-1 thymidine kinase gene to a normal rat, and 1 hr after intravenous administration of F-18 labeled FHBG, a substrate for the tk gene. False color scale indicates increasing levels of tracer uptake in the lungs, demonstrating effective transduction of lung epithelium with the viral kinase (B). Transverse view of the same rat, at the level of the heart. White outlines in both images indicate lung borders.

Figure 1

Copyrighted By Daniel P. Schuster M.D.