In Vivo Imaging System in Gene Therapy

8/14/2019 In Vivo Imaging System in Gene Therapy

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XENOGEN CORPORATION IN VIVO BIOPHOTONIC IMAGING TECHNOLOGIES

Xenogen Corporation has developed

a technology known as biophotonicimaging (3, 5, 26) which allows bio-

logical processes, including gene

expression that is both temporal and

spatially defined (e.g., occurring in

defined tissues and organs within the

animal), to be monitored in live ani-

mals in real-time. Genes encoding spe-

cific luciferase proteins are engineered

into cells (e.g., bacterial pathogens

and cancer cell lines) and animals

(transgenic mice) to enable them toproduce light that can be visualized

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 toxi-

cology (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 biolumines-

cence 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 pro-

duced in a fashion similar to that of the native protein. Bioluminescence can be monitored

OVERVIEW SHEETRNAi In Vivo Applications

Living Image®

Software

IVIS® Imaging System

In vivo biophotonic imaging is offered with the Xenogen

IVIS® Imaging System. Living Image® software controls

the imaging process, and analyzes and archives data.



from cells containing these

luciferases using a light sensi-

tive detector, such as a lumi-

nometer. Xenogen uses the

above approach, but appliesit to monitoring real-time

luciferase expression in living

animals; a technique termed

“in vivo biophotonic imag-

ing.” In the same way that

bioluminescent light is trans-

mitted from cells within the

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 viru-

lence 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


Xenogen In Vivo RNAi Applications of 8

In vivo biophotonic imaging incorporates bioluminescently engineered

Bioware™ cells or microorganisms, as well as LPTA® animal models

genetically engineered to express firefly luciferase.

Tag Cell or Bacteria

Tag GeneIVIS® Imaging System

• Digitize• Quantify• Archive



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) ani-

mal 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 geneexpression 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





Monitoring Viral Delivery of iRNA-Expressing DNA ConstructsIf the experimental approach is to deliver a DNA construct expressing either dual comple-

mentary 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 haveused 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 prob-lematic, 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 andmetastases. 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.



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 represent-

ative mouse. Selected tissues were imaged

ex vivo to confirm in vivo signals.

Intra-cardiac PC-3M-luc injection

Day 7 Day 21 Day 28



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), hepati-tis 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 inves-

tigate 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.

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

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© Xenogen Corporation, 2003. XCAR-1005A. All rights reserved. Trademarks: Xenogen, Discovery in the Living

Organism, Bioware, IVIS, Living Image and LPTA are trademarks and/or trade names of Xenogen Corporation.

Xenogen Corporation, 860 Atlantic Avenue, Alameda, CA 94501, USA Toll Free 877.936.6436Phone 510.291.6100 Fax 510.291.6196 E-mail: [email protected] www.xenogen.com

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36. Zhang, J., X. Tan, C. H. Contag, Y. Lu, D. Guo, S. E. Harris, and J. Q. Feng. 2002. Dissection of

promoter control modules that direct Bmp4 expression in the epithelium-derived components of

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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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