In Vivo Imaging System In Gene Therapy

OVERVIEW SHEET RNAi In Vivo Applications Xenogen Corporation has developed a technology known as biophotonic IVIS® Imaging System imaging (3, 5, 26) which allows biological processes, including gene Living Image® expression that is both temporal and Software spatially defined (e.g., occurring in defined tissues and organs within the animal), to be monitored in live animals in real-time. Genes encoding specific luciferase proteins are engineered into cells (e.g., bacterial pathogens and cancer cell lines) and animals In vivo biophotonic imaging is offered with the Xenogen (transgenic mice) to enable them to IVIS® Imaging System. Living Image® software controls produce light that can be visualized the imaging process, and analyzes and archives data. through the tissues of a live animal using specialized imaging equipment and software designed and built by the company. To date, Xenogen’s technology has been used predominantly to facilitate drug discovery in areas such as infectious disease (9, 10, 14, 27), oncology (4, 7, 25), inflammation and toxicology (6, 36, 37). Recently, this technology has also been used for the assessment of the capability of RNAi molecules to regulate gene expression in live animals (20, 21, 32), enabling a researcher to more rapidly assess whether an RNAi is being delivered to the target tissue to effectively reduce translation of a specific mRNA. This overview gives a scientific approach on how biophotonic imaging can be used to facilitate research and development of RNAi in live animals, and provides an insight into how small RNAi molecules might be better developed as human therapeutics.

Overview of Xenogen Technology Bioluminescence is a biological process by which certain organisms can generate light through an enzyme-mediated reaction. Firefly, glowworm and certain bacteria (commonly associated with fish and squid) are probably the most familiar examples of this phenomenon, all producing visible light. The proteins involved in both firefly and bacterial bioluminescence have been identified and the genes that encode them have been cloned. In both cases, the proteins responsible for bioluminescence are called luciferases. These enzymes generate bioluminescence via a biological reaction in which oxygen and a luciferin substrate react in the presence of a cellular energy source (e.g., ATP) to produce photons of light. The application of these bioluminescent systems to monitor gene expression in cells is now routine in molecular and cellular biology. Typically, the luciferase gene(s) is cloned adjacent to the region of a gene controlling expression (the promoter), such that the luciferase is produced in a fashion similar to that of the native protein. Bioluminescence can be monitored

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from cells containing these luciferases using a light sensitive detector, such as a luminometer. Xenogen uses the above approach, but applies Tag Cell or Bacteria it to monitoring real-time • Digitize luciferase expression in living • Quantify • Archive animals; a technique termed Tag Gene IVIS Imaging System “in vivo biophotonic imaging.” In the same way that In vivo biophotonic imaging incorporates bioluminescently engineered bioluminescent light is transBioware™ cells or microorganisms, as well as LPTA® animal models mitted from cells within the genetically engineered to express firefly luciferase. firefly tail, or bacterial cells within a symbiont (e.g., flashlight fish), so light emitted from bioluminescently engineered cells (e.g., pathogenic bacteria, cancer cells, or transgenic tissue) placed or generated within a small animal (e.g., a mouse or rat) can be detected at the surface (26). Animal tissue will allow light passage to some degree (imagine a flashlight held behind a hand and seeing the red light shining through), and with a suitably sensitive detector (e.g., CCD camera) and image processing software, low levels of light emitted by bioluminescent cells within an animal can be detected, transformed into graphic displays and analyzed. Xenogen has perfected this technique by designing and building its own imaging system, the IVIS® Imaging System, as well as Living Image® analysis software. ®

Typically, the bioluminescent light generated by genetically engineered cells can penetrate 1– 2 cm of tissue making mice ideal subjects to monitor such activity. The location and number of such cells can then be tracked in the live animal. Moreover, the same animal may be imaged multiple times, so allowing the expansion or regression of the disease to be followed (e.g., during infectious disease or oncology studies). Biophotonic imaging is unique in that it can be applied to monitor virtually any biological process in real-time in a live animal, whether that process be the induction of a particular cytokine by the host (e.g., mouse IL-6) in response to an invading pathogen, or a virulence factor induced in a pathogen (e.g., bacterial hemolysin) in response to its invasion of a host. Furthermore, because different luciferases often use different substrates and emit light at different wavelengths, as in the case of firefly and bacterial luciferase, it is possible to monitor two biological events in the same animal at the same time. Thus, in the above example

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it should be possible to monitor both the induction of the hemolysin in the bacteria as it infects the host, and the host’s response to this bacterium with regard to its IL-6 induction. In addition to a large number of infectious disease (9, 10, 14) and oncology (4, 7, 25) animal models that have been developed at Xenogen, an extensive program has also been established for the generation of transgenic animals expressing firefly luciferase, designated as LPTA™ animal models, under the control of different inducible promoters [e.g., inducible nitric oxide synthase promoter, VEGFR2 promoter, rat insulin promoter, heme oxygenase promoter (6, 37) and bone morphogenesis protein 4 promoter (36)]. These LPTA® animal models allow the effects of a particular compound (chemical or biological) to be visualized in the whole animal as that compound is absorbed and metabolized by the different tissues/organs of that animal. Thus, multiple data points can be collected over time and from different regions (tissues/organs) within the same animal.

Background on RNAi Research Since the first successful report of the use of small interfering RNA (siRNA) to silence gene expression in mammalian cells (8), a flood of papers reporting the use of RNA interference (RNAi) to elucidate mammalian gene function has followed. Delivery of synthetic siRNAs to mammalian cells in culture can be achieved using lipophilic agents or electroporation. Alternatively, interfering RNAs can be expressed from a plasmid harbored by the cells of interest, in which pairs of short complimentary RNA molecules (17, 23, 35), or a single inverted small hairpin RNA (shRNA) are stably expressed and used for RNAi gene silencing (1, 22, 30, 35). However, the efficiency of transfection depends on the cell type, as does the ability of a given siRNA to silence a particular gene, making interpretation of RNAi experiments difficult. The use of RNAi in living mice has also been widely reported (2, 12, 18, 20, 21, 28, 29, 31, 32, 34), fueling hope that siRNAs may one day be used to treat human diseases. Again, two strategies for the introduction of RNAi molecules have been used for animal experiments: synthetic siRNA or shRNA delivered directly, or delivery of a plasmid or viral siRNA/shRNA expression cassette that potentially provides a more stable and long lasting delivery of the RNAi species. Although luciferase reporters were used in a number of these studies (18, 20, 21, 32), green fluorescent protein has also proven popular as an alternative reporter (2, 12, 18, 28, 31, 34). However, whereas the use of luciferase has allowed quantitative non-invasive analysis of gene suppression in live animals, studies using GFP as a reporter have required ex vivo tissue extraction or cell rescue and FACS to allow visualization of GFP suppression. Moreover, quantification can only be accurately achieved using Northern analysis, which are time consuming and require sacrifice of the experimental animals. Similarly, detection of RNAi effects on specific host gene suppression (12, 28, 29) have again required FACS, Northern and western analysis of host tissue and cells. Xenogen’s biophotonic imaging technology provides an ideal strategy to non-invasively monitor RNAi in small mammals. In 2002, McCaffrey et al. at Stanford University (20, 21) reported the success of both siRNA and shRNA approaches to reduce luciferase expression in mice following hydrodynamic transfection methods to introduce the RNAi and luciferase

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plasmid. Synthetic siRNA or a plasmid expressing an shRNA designed to target the firefly luciferase gene was monitored in living animals using whole-body imaging. The results of these experiments demonstrate the utility to both track delivery of the siRNA or siRNAexpressing construct and determine efficacy in knocking down the target gene. In vivo animal testing of RNAi for gene knockdown still remains potentially arduous due to a number of factors. Lack of an RNAi effect in vivo could be due to failure of delivery of the siRNA to the target tissue, lack of si/shRNA expression from a construct, lack of response by the target, or lack of specificity of the siRNA for its target. Xenogen offers a number of approaches and tools to facilitate the evaluation of RNAi in vivo and to assess these factors.

Testing of siRNA in LPTA® Animal Models Expressing Luciferase Xenogen has created a variety of LPTA® animal models expressing luciferase in specific tissues such as liver, kidney, stomach, intestine and pancreatic islets. These models are useful for following disease progression and/or response to treatment. For example, the iNos-luc LPTA® mouse Control LPS+IFN-γ model uses a portion of the murine iNos promoter to drive luciferase expression. This reporter is induced in hepatic Kuppfer cells when lipopolysaccharide is injected (Figure 1). Targeting siRNA or shRNA against luciferase for these existing LPTA® animal models would be useful to assess delivery of the iRNA or construct into tissues in which luciferase is expressed. iNos-luc LPTA™ Animal Model Figure 1. Induction of iNos-luc reporter by LPS. Male iNos-luc mice injected with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN) and imaged 6 hours later show a strong induction of the luciferase signal in liver Kuppfer cells.

Custom Transgenic Model Development In addition to Xenogen’s currently developed LPTA® animal models, custom transgenic models can be developed at Xenogen to test targeting and knockdown efficiency against a specific target. A custom transgenic model could be built by Xenogen that uses the promoter for the gene of interest, with the luciferase coding sequence fused to the 5´ UTR, or as a fusion to a region of the open reading frame of the target determined to be suitable for RNAi knockdown. Studies have indicated that siRNA targeted to the first 100bp of the coding region of a gene are most successful, suggesting that short fusions of these same regions to the N-terminal of luciferase will result in a functional luciferase that could be switched off by the appropriate siRNA. Either human or mouse gene sequence could be used, but using a transgenic containing a construct with the human gene sequence fused to the luciferase gene would allow testing, in vivo, of the efficiency for knockdown of the human gene.

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Monitoring Viral Delivery of iRNA-Expressing DNA Constructs If the experimental approach is to deliver a DNA construct expressing either dual complementary siRNAs or shRNA to a target tissue, then the luciferase gene can be incorporated to express luciferase driven by either a constitutive promoter or a tissue specific promoter. There are several examples of adenoviral delivery of constructs for gene therapy that have used luciferase reporters for this purpose. For example Laxman et al. (16), have shown the feasibility of tracing delivery of a therapeutic adeno-associated virus construct into a brain tumor in mouse using a firefly luciferase reporter. Similarly, Lipshutz et al. (19) injected an adeno-associated virus into the peritoneal cavity of 15-day-old fetuses and were able to show that the luciferase reporter was still expressed in the peritoneum in mice up to 18 months of age. Finally, Tsui et al. (32) delivered a lentiviral vector expressing either human factor IX or a luciferase reporter by intravenous injection and were able to follow the kinetics of gene transfer in adult mice. The clear advantage of this approach is that one can follow the time course of delivery and persistence of the vector in the target tissue.

RNAi Specificity For Target Inactivation Identification of suitable sequences within a specific gene for RNAi targeting remains problematic, but can often be optimized in mammalian tissue culture experiments. However, it has been shown that siRNAs can be ineffective in some cell types compared to others. The use of whole animal experiments to identify the effects of specific siRNAs within tissues would provide the ultimate confirmation of the specificity and activity of an RNAi as a potential therapeutic. The application of custom LPTA® animal models with fusions of luciferase to target sequences may provide such an assay.

Evaluating RNAi Treatment of Tumors Xenogen has developed a set of human tumor cell lines, constitutively labeled with luciferase, and termed Bioware™ cells, that are used for non-invasively monitoring xenograft growth and metastases. With luciferase-labeled cell lines one can more easily follow the early stages of tumor growth, and also detect metastases (Figure 2). For the development of RNAi therapies against tumors, these in vivo systems would be an ideal approach for evaluating efficacy in animal models. Day 7

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Figure 2. Metastatic model with injection of PC-3M-luc prostate tumor cell line. PC-3M-luc cells were injected into the left ventricle of male athymic mice, and ventral images are shown for a representative mouse. Selected tissues were imaged ex vivo to confirm in vivo signals.

Intra-cardiac PC-3M-luc injection

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Antiviral and Antimicrobial Treatment with RNAi A number of publications have reported the use of siRNA to interfere with and block viral replication and viral RNA transcription in cultured mammalian cells. Transfection of siRNA duplexes into cell lines has been shown to inhibit human immunodeficiency (13, 17), hepatitis C (15, 24, 33) and influenza (11) virus replication for several days. Further, McCaffrey et al. [see above, (20, 21)] used in vivo imaging to show that siRNA targeting a region of a hepatitis C virus (HCV) fused to luciferase could be used to reduce production of the chimeric HCV-luciferase protein by over 75%. The application of animal models to investigate viral infections has been limited by the ability of the animal host to be infected by the viral pathogen of interest. Should RNAi prove to be a suitable approach for treating viral infections, biophotonic imaging may provide a powerful tool to test such therapies. As suggested above, chimeric fusions of viral proteins and luciferase could be made, and then the efficacy of the siRNA tested by biophotonic imaging.

Summary Applications of Xenogen biophotonic imaging for RNAi research and development: m In vivo target validation for drug discovery in all therapeutic areas m Testing RNAi therapeutic approaches in vivo m Tracking and monitoring siRNA and shRNA delivery in vivo

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Note: For LPTA® animal model lines CYP3a11, CYP3A4 rat, Epx, Vegfr2 and Vegf: these product lines and their use are claimed by pending U.S. and foreign patent applications owned by Xenogen Corporation. LPTA® animal model lines and certain Bioware™ cell lines contain a luciferase gene provided under a license from Promega Corporation. Under the terms of that license, the use of these products and derivatives thereof is strictly limited to that of a research reagent. No right to use these products for any diagnostic, therapeutic, or commercial application will be conveyed to the customer of these products. In vivo imaging in mammals is covered by one or more U.S. and foreign patents controlled by Xenogen Corporation, including the following: U.S. patent numbers 6,217,847 and 5,650,135 and European Union patent number 0861093. A license from Xenogen Corporation is required to practice under these patents.

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