Current Gene Therapy (v.12, #1)

One of the reasons why gene medicines have yet to reach the stage of licensed use in the West may be the lack of available robust methods to accurately monitor events post vector administration to patients. Traditional biopsy procedures have obvious limitations in assessing vector trafficking and transgene expression kinetics and levels, not least the impracticality of taking biopsy samples at multiple evaluation points or from every organ of interest. In response to the first gene therapy death, the NIH Recombinant DNA Advisory Committee (RAC) called for better assays for measuring transgene expression in cells and tissues [1]. Non-invasive imaging modalities are therefore under heavy investigation in recent years. By definition, molecular imaging (MI) techniques directly or indirectly monitor and record the spatio-temporal distribution of molecular and cellular processes for biochemical, biological, diagnostic or therapeutic applications (Radiological Society of North America and the Society of Nuclear Medicine) [2]. Use of MI stands to facilitate acquisition of the required data at various phases throughout the clinical protocol, in terms of both a) assessment of the location, magnitude and duration of transgene expression and b) monitoring of responses to intervention. Since the first conception of the radiotracer principle by George de Hevesy described in Nature in 1935 [3], a basis has existed for development of MI technology. Molecular imaging combines various disciplines, including cell biology, molecular biology, chemistry, physics and medicine. Only within the last decade have parallel developments in in vivo imaging technologies enabled routine use of MI modalities in pre-clinical and clinical settings. The reviews contained in this issue are not exhaustive with respect to the range of imaging technologies and reporter strategies pertinent to gene & cell therapy. The modalities of imaging technology used to detect reporter gene expression focused on in this issue include Optical, SPECT and PET, for imaging of various radio-tracers or light emissions. These represent the most widely used imaging modalities. Other clinical imaging technologies including MRI/MRS, exploiting a range of reporter genes are recently under investigation (for review, see [4]). However, the extent of this research in the context of gene therapy is limited, and the reviews presented in this issue focus on more established technologies. The various modalities differ in a number of key aspects, such as sensitivity; resolution (both spatial (μm - cm) and temporal (milliseconds to hours)); tomographic potential; depth penetration; availability of imaging probes; throughput level; cost; ease of operation; and potential for clinical transfer [4]. In the context of gene therapy, the most significant clinical progress has been made using PET and SPECT. While the sensitivity of PET imaging (see Collins et al. review) is high and the speed of imaging is relatively rapid (minutes), these techniques lack micrometer spatial resolution (1-2 mm with micro-PET). An alternative approach to PET is SPECT imaging (see review by Carlson et al.). While the sensitivity of the single-photon system can be two orders of magnitude less than the PET systems, the required radionuclides and hardware are more available. While not discussed in detail here, the advantage of MRI for potential imaging of gene expression is high 3D spatial resolution (tens of mm range); but sensitivity is low, requiring high concentrations of tracer and long exposure times. Overall, each method is not without its limitations, and there is certainly a need to develop protocols with improved signal sensitivity and resolution. Disadvantages of a given technique may be complemented through combination with another; e.g to provide highly sensitive molecular information (using PET or optical) with high spatial resolution (through MRI). Real-time imaging will be particularly beneficial to specify treatments to individual patients, particularly in the context of multigenic diseases such as cancer. Active collaboration between biological scientists, physicists, chemists and clinicians is required to drive the field forward to clinical use. The state-of-the-art as described here indicates that the translational jump for the current preclinical technologies may not be high.

Integral to the development of all gene therapy technologies is the ability to monitor gene delivery, in terms of distribution, levels and kinetics of vector transgene expression. This can be achieved to some extent at the preclinical level through use of traditional ex vivo analytical methods, but these hold several drawbacks, not least the requirement for death of experimental subjects for such end-point assays. Real-time in vivo analysis of reporter gene expression empowers the investigator with the ability to non-invasively assess gene delivery over time, as well as host responses to vector administration and therapeutic interventions. While there exist several technologies for such small animal monitoring, imaging of light emission from luminescent or fluorescent reporters has become the mainstay of preclinical imaging for gene therapy research. Optical imaging strategies represent powerful yet cost-efficient and convenient systems compared with alternative methods. Through tagging of vector and/or cells or interest with suitable reporter genes, both vector and host responses can be assessed in rapid, high-throughput analyses, providing spatial, temporal and quantitative read-out, without the need for radioactivity. In this review, we discuss the current state-of-the-art for optical technologies, describe related approaches employed in gene therapy research for a wide range of diseases, and outline the potential for this imaging modality in the progression of gene therapy as a medicine.

Bacterial production of visible light is a natural phenomenon occurring in marine (Vibrio and Photobacterium) and terrestrial (Photorhabdus) species. The mechanism underpinning light production in these organisms is similar and involves the oxidation of an aldehyde substrate in a reaction catalysed by the bacterial luciferase enzyme. The genes encoding the luciferase and a fatty acid reductase complex which synthesizes the substrate are contained in a single operon (the lux operon). This provides a useful reporter system as cloning the operon into a recipient host bacterium will generate visible light without the requirement to add exogenous substrate. The light can be detected in vivo in the living animal using a sensitive detection system and is therefore ideally suited to bioluminescence imaging protocols. The system has therefore been widely used to track bacteria during infection or colonisation of the host. As bacteria are currently being examined as bactofection vectors for gene delivery, particularly to tumour tissue, the use of bioluminescence imaging offers a powerful means to investigate vector amplification in situ. The implications of this technology for bacterial localization, tumour targeting and gene transfer (bactofection) studies are discussed.

PET Imaging for Gene ' Cell Therapy by Sara A. Collins (20-32).
As the interest in gene therapy increases, the development of an efficient and reliable means to monitor gene delivery and expression in patients is becoming more important. An ideal imaging modality would be non-invasive, allowing for repeated imaging, thus validating stages subsequent to vector administration and allowing for the improvement of clinical protocols. Positron Emission Tomography (PET) has been employed for some time in clinical imaging and has in more recent years been adapted to enable imaging in small animal models, including gene therapy models for a range of diseases. PET imaging is based on the detection of trace quantities of positron-emitting molecular probe within cells postadministration, permitting imaging of target molecules in vivo, and numerous tracers have been developed for a wide range of applications, including imaging of reporter gene activity. Use of radiolabelled substrates that interact with specific transgene proteins, has identified a number of reporter genes that are suitable for imaging vector mediated gene delivery and expression in both pre-clinical and clinical situations. These reporter genes enable non-invasive analysis of the location, level and kinetics of transgene activity. Among the various imaging modalities in existence, the PET approach displays arguably the optimum characteristics in terms of sensitivity and quantitation for in vivo gene expression measurements. Given the existing availability of PET scanning equipment and expertise in hospitals, this imaging modality represents the most clinically applicable means of analysing gene therapy in patients. This review outlines the principles of PET imaging in the context of gene and cell therapy at both pre-clinical and clinical levels, comparing PET with other relevant modalities, and describes the progress to date in this field.

Preclinical and clinical tomographic imaging systems increasingly are being utilized for non-invasive imaging of reporter gene products to reveal the distribution of molecular therapeutics within living subjects. Reporter gene and probe combinations can be employed to monitor vectors for gene, viral, and cell-based therapies. There are several reporter systems available; however, those employing radionuclides for positron emission tomography (PET) or singlephoton emission computed tomography (SPECT) offer the highest sensitivity and the greatest promise for deep tissue imaging in humans. Within the category of radionuclide reporters, the thyroidal sodium iodide symporter (NIS) has emerged as one of the most promising for preclinical and translational research. NIS has been incorporated into a remarkable variety of viral and non-viral vectors in which its functionality is conveniently determined by in vitro iodide uptake assays prior to live animal imaging. This review on the NIS reporter will focus on 1) differences between endogenous NIS and heterologously-expressed NIS, 2) qualitative or comparative use of NIS as an imaging reporter in preclinical and translational gene therapy, oncolytic viral therapy, and cell trafficking research, and 3) use of NIS as an absolute quantitative reporter.

Phenylketonuria (PKU) is one of the most common inborn errors of metabolism and is due to a deficit of phenylalanine hydroxylase, the enzyme that converts phenylalanine (Phe) into tyrosine (Tyr). The resultant hyperphenylalaninemia (HPA) leads to severe neurological impairment, whose pathogenesis has not been entirely elucidated. Treatment of PKU consists essentially in lifelong protein restriction and, in mild cases, in tetrahydrobiopterin supplementation. However, compliance to both strategies, particularly to the long-term diet, is low and therefore other therapies are desirable. We explored a gene therapy approach aimed at long-term correction of the pathologic phenotype of BTBR-PahEnu2 mice, a mouse model of PKU. To this aim, we developed a helper-dependent adenoviral (HD-Ad) vector expressing phenylalanine hydroxylase and administered it to 3-week-old PKU mice. This resulted in complete normalization of Phe and Tyr levels and reversal of coat hypopigmentation that lasted throughout the observation period of six months. The spatial learning deficits observed in PKU mice were also reversed and hippocampus levels of the N-methyl-D-Aspartate and 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid receptor subunits returned to normal. Long-term potentiation, which is impaired in PKU mice, was also restored by treatment. Therefore, HD-Ad vector-mediated gene therapy is a promising approach to PKU treatment.

The severe combined immunodeficiency caused by the absence of adenosine deaminase (SCID-ADA) was the first monogenic disorder for which gene therapy was developed. Over 30 patients have been treated worldwide using the current protocols, and most of them have experienced clinical benefit; importantly, in the absence of any vector-related complications. In this document, we review the progress made so far in the development and establishment of gene therapy as an alternative form of treatment for ADA-SCID patients.