WO1998033935A1

WO1998033935A1 - Method for detecting exogenous gene expression

Info

Publication number: WO1998033935A1
Application number: PCT/IB1998/000123
Authority: WO
Inventors: Leonard I. Wiebe; Edward E. Knaus; Kevin W. Morin; Alexander Mcewan
Original assignee: The University Of Alberta
Priority date: 1997-01-31
Filing date: 1998-01-30
Publication date: 1998-08-06
Also published as: AU5676398A

Abstract

Disclosed are methods of determining the expression of an exogenous reporter enzyme gene in a target cell by introducing into the cell a construct comprising a reporter enzyme gene coding for a reporter enzyme which has a reductive function, thereafter introducing to the target cell a labelled substrate which is specific for the reporter enzyme, wherein any reporter enzyme present in the target cell reduces the labelled substrate to produce a labelled product which accumulates in the target cell, and detecting the labelled product to determine the expression of the reporter enzyme gene in the target cell. Use of a labelled substrate to determine the expression of an exogenous reporter enzyme gene which has a reductive function is also disclosed, as is a method for monitoring the presence and/or the amount of expression of an exogenous construct.

Description

METHOD FOR DETECTING EXOGENOUS GENE EXPRESSION

FIELD OF THE INVENTION

This invention relates to a diagnostic method for detecting the

expression of exogenous genes in vitro or in vivo. Preferably, the exogenous

gene is a gene which codes for a reductive enzyme.

BACKGROUND OF THE INVENTION

The advent of molecular biology and new efficient means of gene

transfer have provided new treatment strategies which are based on transfer

of genetic material. Historically, gene therapy has referred to the transfer of a

normal gene to correct an aberrant gene defect. This traditional view of

corrective gene transfer is applicable to a variety of genetic abnormalities.

Introducing genes into cells that are not present or adequately expressed in

these cells is a general strategy that encompasses most experimental

approaches for gene therapy. An ideal gene therapy protocol would involve

efficient delivery of the gene of interest to a tissue requiring the gene product.

Following delivery, the gene should be expressed at a level that yields a

therapeutic effect. The desired level of gene expression should continue for

a period of time that should be sufficient for beneficial effects to the recipient.

Most often, in corrective gene therapy, it is desirable for the delivered

therapeutic gene to be expressed as long as possible.

Gene transfer based on retroviral vector delivery vehicles is a useful

method of introducing genes into cells. For example, adenosine deaminase

(ADA) deficiency has been treated by in vitro retroviral gene transfer of the

ADA gene to human T lymphocytes (Anderson, 1992). Similarly, neoplasms are efficiently transduced with retroviral vectors carrying cytokine or drug sensitivity genes (Morsy et al., 1993; Mullen, 1994). Although gene delivery by retroviral vectors often gives long term expression of the transgene, it can only transduce tissue that is replicating. Therefore, tissue that is not actively replicating is incapable of integrating the genes carried on the vector.

In contrast, adenoviral vectors are highly efficient viral vectors that are capable of delivering genes to non-replicating cells. For example, the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been efficiently transferred to airway epithelium in patients with cystic fibrosis (Rosenfeld et al., 1992). In cancer gene therapy, adenoviral vectors have been shown to be useful for the delivery of prodrug-activating suicide genes like herpes simplex virus type-1 thymidine kinase (Chen et al., 1994). However, adenoviral vectors evoke inflammatory immune responses that limit

the duration of transgene expression. The transient nature of expression is also a consequence of non-integration since the viral genome is maintained as an extrachromosomal episome. Therefore, although extremely efficiently delivered, adenoviral vectors suffer from a brief duration of action (gene expression).

Plasmid vectors are also capable of delivering a therapeutic gene to

cells. However, the process of delivery is vastly inefficient compared to viral vector systems and therefore considerable effort has been directed towards improved methods of delivery. Currently, cationic liposomes are the most popular reagents used with plasmid DNA to facilitate entry into the cell. Following entry and migration to the nucleus, plasmid vectors can express the therapeutic gene product for a limited time. Like adenoviruses, plasmids are generally not integrated into the host genome and therefore gene

expression is usually transient.

Detecting gene expression during in vivo gene therapy is a critical issue for laboratory and clinical investigators. Unfortunately, the most

sensitive and reliable techniques to date for detecting and quantitating in vivo gene transfer and expression have been invasive. To date, these invasive techniques have been invaluable for evaluating extent of gene delivery and expression. Vector design and construction has often exploited the use of common reporter systems that invariably involve removal of host tissue for histochemical evaluation. In principle, generic reporter constructs encode for gene products that enzymatically alter exogenous substrates to easily detectable species. For example, beta-galactosidase gene transfer and expression can be detected in vitro by histochemical staining with X-gal (Alam and Cook, 1990). The firefly luciferase gene is frequently employed as a reporter gene and its gene product is detected by luminescence assay with luciferin (Nguyen et al., 1988). Other reporter genes include chloramphenicol acetyltransferase, green fluorescent protein and alkaline phosphatase (Gorman er a/., 1982; Laimins, 1984; Berger et al., 1988). Each of these reporter gene systems require invasive biopsy procedures for evaluation of

gene expression.

Tissue-selective metabolic processing is a fundamental tenet for preferential radiopharmaceutical localization in vivo. Scintigraphic imaging techniques used routinely in nuclear medicine have potential applications in gene therapy as a means of non-invasively detecting gene expression. The development of an efficient and highly selective reporter gene and ligand system is essential for non-invasive imaging studies. Similarly, compounds

that contain NMR-sensitive labels can be used with NMR imaging and spectroscopy methods to image and characterize the bio-distribution of the NMR-sensitive label that accumulates in the cell through the action of the protein expressed by the transferred gene.

Nitroimidazole radiopharmaceuticals localize in hypoxic tissue due to preferential hypoxia-dependent nitro-reduction (Parliment et al., 1992; Urtasun et al. , 1996; Groshar et al., 1993 ). The mechanisms responsible for nitroimidazole binding in hypoxic tissue have been studied extensively. For example, misonidazole has been shown to bind to cellular macromolecules in hypoxic tissue due to selective reversible single electron reduction and subsequent generation of reactive intermediates such as the hydroxylamino derivative which is capable of covalent binding (Chapman ,1984). The binding of nitroimidazole radiopharmaceuticals in hypoxic tissue allows the use of non-invasive scintigraphic imaging techniques to detect the hypoxic tissue. Selective uptake and accumulation of nitroimidazole radiopharmaceuticals in hypoxic tissue allows for specific imaging since these compounds are labelled with radioactive isotopes that can be readily detected with nuclear medicine techniques such as planar scintigraphy, single photon emission computed tomography (SPECT) and positron emission tomography (PET). Other radiopharmaceuticals may localize in hypoxia as a result of change of oxidation state of a metal ion component, resulting in decreased solubility and consequently, accumulation in hypoxic cells, as shown in Scheme 1 below.

(not reversible by 0₂)

Reduction Products

Hypoxic Cells (Nitroso, Hydroxylamino, Amino)

Oi

Scheme 1. Misonidazole Reduction in Hypoxic cells.

The nfnB gene of Escherichia coli, which encodes for a nitroreductase, has recently been cloned and the product has been identified as having potential utility as a prodrug-activating enzyme (Michael et a/., 1994). This nitroreductase can reduce nitro-functionalities on aromatic compounds that are resistant to reduction by human reductases (Anlezark et al., 1995).

Consequently, compounds have been designed and synthesized that are

selectively reduced by E. coli nitroreductase to yield cytotoxic derivatives (Knox et al. , 1992). In particular, nitroaromatics have been shown to be selectively reduced to bifunctional alkylating agents with considerable cytotoxicity. E. coli nitroreductase reduces nitroaromatic compounds through a sequential 2-electron reduction mechanism, to a reactive hydroxylamino species (Michael et ai, 1994). Since nitroaromatic radiopharmaceuticals

undergo a similar reduction in hypoxic tissue, these diagnostic agents would seem to be promising markers of tissues that express E. coli. nitroreductase, since cell-specific binding or accumulation can occur after reduction to reactive intermediates. The present inventors have discovered that E. coli. nitroreductase, a completely foreign gene to mammalian organisms, can serve as a reporter gene product with subsequent detection with nitroimidazole radiopharmaceuticals when the nitroreductase gene is introduced into non-hypoxic cells using gene transfer techniques.

There is a need for a diagnostic method which may be used in conjunction with gene therapy techniques to monitor the transfer of a gene construct in vivo and non-invasively. This diagnostic method would function like a reporter gene system, but would allow non-invasive biochemical evalution. There is a need to identify the genes and gene products necessary for performing this diagnostic method. In addition, there is a need to identify compounds possessing specific properties which are suitable for use in performing this diagnostic method.

SUMMARY OF THE INVENTION The present invention provides for diagnostic methods which may be

used to detect whether an exogenous gene has been incorporated into the

target cells and is being expressed therein. The invention also provides for the use of labelled compounds in performing these methods.

The invention is applicable to populations of cells into which a gene construct has been transferred. In a preferred embodiment, the construct includes a reporter enzyme which is not naturally occurring in the target cells. Preferably, a substrate reacts specifically with the reporter enzyme, enters cells readily and produces a labelled product which accumulates in those cells or in the vicinity of the reporter enzyme, thus facilitating detection

and mapping of the labelled product.

The invention thus includes a method for monitoring the transfer of a construct and the expression of reporter enzyme in populations of cells (e.g., in vitro or in vivo) or an organism (i.e., in vivo).

The invention includes the use of a reporter enzyme gene in a construct used in gene delivery or gene therapy; and the use of labelled substrates to detect and map the functional expression of the reporter enzyme using nuclear medicine techniques or magnetic resonance techniques.

The method can also be used to determine the stability of the reporter enzyme gene in the transformed cells.

Subsequent to the transfer of the construct to the target cells (which can be less than 24 hours, 1-30 days, 1-12 months or even 1-5 years later), a

) labelled substrate is administered to the cells or organism in an amount sufficient to produce a detectable or measurable signal. For organisms, nuclear medicine images or nuclear magnetic images are then recorded and analyzed using standard nuclear medicine or magnetic resonance

techniques. The labelled product accumulates within those cells in which the

reporter enzyme has been expressed.

The invention comprises the use labelled substrates in performing this

diagnostic method.

The present invention can be used to develop targeted delivery systems and to verify the specificity or accuracy of the targeting system and to determine the stability of the construct expressing the reporter enzyme in

the targeted cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The term "reporter enzyme" herein refers to a protein that initiates a chemical transformation of the labelled substrate. Reporter enzymes can, in principle, be an enzyme selected from any eucaryotic or procaryotic cells. Preferred reporter enzymes are those which have a reductive function, and include, but are not limited to a nitroreductase (e.g., E. coli nitroreductase, although the use of other nitroreductases is also within the scope of the invention), Cytochrome P450 reductase, DT-diaphorase or Xanthine Oxidase. More preferred reporter enzymes are a nitroreductase and Cytochrome P450 reductase.

The term "substrate" herein refers to a compound whose chemical transformation is catalyzed by the reporter enzyme. The substrate varies with the reporter enzyme. The substrate is labeled with a radiolabel or an NMR- sensitive label so that it can be detected using nuclear medicine techniques or magnetic resonance techniques well known to those of ordinary skill in the

123 124 125 131 75 art. Suitalbe radiolabels include, but are not limited to, I, I, I, I, Br, ⁷⁶Br , ¹⁸F, ^Cu, ⁶²Cu or ""Tc. Suitable NMR-sensitive labels include, but are

not limited to, ¹⁹F, ¹³C, ³¹P or ¹¹B. Other NMR-sensitive labels can be found in Koutcher et al. I and II, 1984; Tyler et al., 1984). A substrate is chosen based on the properties of the enzymatic product and the specificity for the reporter enzyme. The labelled product accumulates in cells which express the reporter enzyme, or near the expressed enzyme. When the reporter enzyme has bio-reductive properties, prefered substrates for these reporter enzyme include compounds represented in Formulas 1 through 10.

The term "vector" used herein means any DNA or RNA material capable of transferring genetic material into a host cell. It may be linear or circular in topology and includes, but is not limited to plasmids, bacteriophages, retrovirus, adenoviral and DNA viruses. The vector can be introduced into cells using known gene delivery methods with gene delivery compositions known to someone skilled in the art. The term "construct" herein refers to a vector containing one or more copies of a reporter enzyme operably linked to regulatory genes for the functional expression of this enzyme. This construct may include one or more other structural genes and operably linked regulatory genes. In a preferred embodiment, the construct includes other structural genes useful for gene therapy (i.e., therapeutic genes). These other structural genes can be unknown to the genome of the target cells, or can be present in the

genome in inactive, overactive, or otherwise mutated or defective form. The therapeutic genes would be selected to treat the disease caused by the defective gene, or to introduce a therapeutically useful property into the cells which was otherwise unknown in the cell genome. Upon knowing the defect

in the cell genome, a worker of ordinary skill in the art would be able to select a therapeutically useful gene for treating the disease caused by the defect

without undue experimentation.

The label used to label the substrate is preferably a radiolabel, but any other form of labelling which facilitates detection of the labelled product using non-invasive procedures, for example by magnetic resonance techniques, may also be suitable. When the reporter enzyme is a gene coding for a nitroreductase or Cytochrome P450 reductase, the preferred labelled

substrates include compounds represented by Formulas 1 through 10, as follow, or pharmaceutically acceptable salts thereof:

Formula 1 wherein X is a radiolabel or an NMR-sensitive label;

Formula 2 wherein X is a radiolabel or an NMR-sensitive label;

Formula 3 wherein X is a radiolabel or an NMR-sensitive label;

Formula 4 wherein the "^mTc label can be replaced by a ⁶⁷Ga or ¹¹¹ln label or an NMR- sensitive label can be incorportated at the position indicated by the (^*);

Formula 5 wherein the ^rømTc label can be replaced by a ⁶⁷Ga or ¹¹¹ln label or an NMR- sensitive label can be incorportated at the position indicated by the (^*);

Formula 6 wherein the """Tc label can be replaced by a ⁶⁷Ga or ¹¹¹ln label or an NMR- sensitive label can be incorportated at the position indicated by the (^*);

Formula 7 wherein the ""Tc label can be replaced by a ⁶⁷Ga or ¹¹¹ln label or an NMR- sensitive label can be incorportated at the position indicated by the (*);

Formula 8 wherein Cu is ^KCu or ^MCu and Cu can be replaced by a ^Ga or ¹¹¹ln label or an NMR-sensitive label can be incorportated at the position indicated by the

(^*);

Formula 9

wherein X is a radiolabel or an NMR-sensitive label;

Formula 10 wherein X is a radiolabel or an NMR-sensitive label.

The present invention includes the use of a reporter enzyme gene in a construct used in gene delivery or gene therapy; and labelled substrates to detect and map the functional expression of the reporter enzyme using

nuclear medicine or magnetic resonance techniques. The following non-

limiting example elucidates the invention. Example

A construct containing one or more nitroreductase genes or other reductase genes such as Cytochrome P450 reductase is prepared according to standard biotechnology techniques. See Sambrook et al., Molecular

Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor Laboratory Press 1989), the entire contents of which are hereby incorporated by reference. The construct can also contain other structural genes useful for gene therapy. In these cases, the reporter enzyme functions as a reporter in the target cells where gene transfer and expression has occurred. However, a construct can also be used where the nitroreductase or other reductase gene is the only gene in the construct. The construct is deliberately transferred, transduced or transfected into the target cells or tissues by standard gene delivery techniques which would be known to one of ordinary skill in the art, such as liposome delivery or by viral vectors. Examples of gene transfer techniques which would be appropriate are included in Culver (1994) and references therein and/or other current molecular biology references.

Subsequent to the introduction of the construct into the target cells, the labelled substrate is administered. An example of a suitable route of

administration when the method of the invention is performed in vivo is by intravenous injection or infusion. The compounds represented in formulas 1- 10 are examples of substrates that can be used to detect the nitroreductase or other reductase gene expression. However, other substrates of related structure can be used for this purpose. The labelled substrate interacts selectively with the expressed reporter enzyme to produce the labelled product. It is expected that not every transferred reporter enzyme gene

and/or other structural genes in the construct is being expressed. Some imported genes may be actively expressed in some cells but not in others. As a result, the labelled product is produced only within those cells in which the reporter enzyme has actually been expressed by the transferred reporter enzyme gene. The labelled product may then be detected in order to monitor the transfer of the reporter enzyme gene in the cells.

The labelled substrate is selected to interact selectively with the

reporter enzyme expressed by the reporter enzyme gene in order to produce a labelled product which accumulates in the target cells in which the reporter enzyme has been expressed. Thus, a preferential accumulation of the labelled product occurs in the reporter enzyme-expressing cells as compared to cells which either do not include the transferred reporter enzyme gene or which include a dormant transferred reporter enzyme gene. This accumulation permits the specific detection of those cells which express the reporter enzyme. Modification of the labelled substrate, such as nitro- reduction to reactive intermediates or metal-center reduction to insoluble species, occurs in the presence of the reporter enzyme, which results in the formation of the labelled product inside the target cell. The diagnostic use and method may be used both in vitro and in vivo to monitor the transfer of the enzyme reporter gene throughout the population of target cells. More specifically, the method may be useful to determine the location or site, the extent and the kinetics of the transfer of the enzyme reporter gene throughout the cells. The diagnostic method may be used in clinical studies to assess treatment efficacy or to determine the stability of the

construct expressing the reporter enzyme and/or other structural genes in the target cells. Stability is determined by repeating the imaging procedure (i.e., the administration of labelled substrate and subsequent detection of labelled product) at least once and comparing the results to determine if expression of the reporter enzyme has changed over time. If expression has decreased or has stopped completely, it can be concluded that the construct is unstable. Nuclear medicine technology or magnetic resonance technology are employed for the detection method. The type of technology employed is dependent on the selected label. Radiolabels can be detected with a suitable technique such as planar scintigraphy, single photon emission computed tomography (SPECT) or positron emission tomography (PET). Substrates labelled with technetium 99m are ideally suited for planar scintigraphy or SPECT imaging. All compounds represented by formulas 1-10 can be detected when labelled with an NMR-sensitive label. References on imaging and spectroscopy applications in drug biodistribution and metabolism are as follows: Maxwell et al., 1991 ; Kristjansen et al. 1993; Pederson et al. 1994.

In summary, cells or organisms undergoing nitroreductase or other reductase gene transfer to target cells can be assessed for the extent of gene transfer and expression using the detection of labelled products. When the method of the invention is used in vivo, the substrates described herein are administered to an organism in a suitable manner that will allow distribution to the entire organism. Following a suitable period of time for localization of the

labelled substrate, an imaging or detection procedure is performed that allows for the non-invasive visuallization of cells or tissue expressing the nitroreductase or other reductase enzymes. Those skilled in the art of interpreting images can ascertain the extent of gene transfer and expression

after administration of labelled compounds that localize specifically in the nitroreductase or other reductase-expressing tissue or cells. References:

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Anderson W. F. Science 1992, 256, 808-813.

Anlezark, G. M.; Melton, R. G.; Sherwood, R. F.; Wilson, W. R.; Denny, W. A.;

Palmer, B. D.; Knox, R. J.; Friedlos, F.; Williams, A. Biochem. Pharmacol. 1995,

50, 609-618.

Berger, J.; Hauber, J.; Hauber, R.; Geiger, R.; Cullen, B.R. 1988, 66, 1-10.

Chapman, J.D. Cancer, 1984, 54, 2441-2449

Chen, S. H.; Shine, H. D.; Goodman, J. C; Grossman, R. G.; Woo, S. L. Proc.

Natl. Acad. Sci. USA 1994, 91, 3054-3057.

Culver, K.W. . Gene Therapy. A Hand for Physicians, 1994.

Gorman, CM.; Moffar. L.F.; Howard, B.H. Mol. Cell Biol. 1982, 2, 1044-1051.

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of determining the expression of an exogenous reporter enzyme gene in a target cell, comprising introducing into a target cell a construct comprising a reporter enzyme gene coding for a reporter enzyme which has a reductive function; thereafter introducing to the target cell a labelled substrate which is specific for the reporter enzyme, wherein any reporter enzyme present in the target cell reduces the labelled substrate to produce a labelled product which accumulates in the target cell; and detecting the labelled product to determine the expression of the reporter enzyme gene in the target cell.

2. Use of a labelled substrate to determine the expression in a target cell of an exogenous reporter enzyme gene which has a reductive function, characterized in that the labelled substrate is introduced to the target cell, wherein any reporter enzyme present in the target cell reduces the labelled substrate to produce a labelled product which accumulates in the target cell, and the labelled product is detected to determine the expression of the reporter enzyme gene in the target cell.

3. A method for monitoring the presence and/or the amount of expression in a target cell of an exogenous construct comprising a reporter enzyme gene coding for a reporter enzyme which has a reductive function, the method comprising

(a) introducing to the target cell a labelled substrate which is specific for the reporter enzyme, wherein any reporter enzyme present in the target cell reduces the labelled substrate to produce a labelled product which accumulates in the target cell;

(b) detecting the labelled product to determine the presence and/or the amount of expression of the reporter enzyme gene in the target cell; (c) repeating steps (a) and (b) at least once; and (d) comparing the first detecting step and any repeated detecting steps to monitor the presence and/or the amount of expression of the exogenous construct.

4. Use of a labelled substrate to monitor the transfer of a reporter enzyme gene that has a reductive function throughout a population of cells, by administering to the cells the labelled compound so that the labelled substrate interacts selectively with the reporter enzyme expressed by the transferred reporter enzyme gene to produce a labelled product and then detecting the labelled product, wherein the labelled substrate is selected to interact selectively with the reporter enzyme expressed by the transfered reporter enzyme gene such that the labelled product accumulates within those of the cells in which the reporter enzyme has been expressed by the transferred reporter enzyme gene.

5. A method for monitoring the transfer of a reporter enzyme gene throughout a population of cells, comprising the following steps:

(a) administering to the cells a labelled substrate so that the labelled substrate interacts selectively with a reporter enzyme that has reductive function expressed by the transferred reporter enzyme gene to produce a labelled reduced product; and (b) detecting the labelled reduced product; wherein the labelled substrate is selected to interact selectively with the reporter enzyme expressed by the reporter enzyme gene such that the labelled product accumulates within those of the cells in which the reporter enzyme has been expressed by the transferred reporter enzyme gene.

6. The method as claimed in claim 5, wherein the labelled substrate is labelled with a radiolabel or an NMR-sensitive label.

7. The method as claimed in claim 5, wherein the transferred reporter enzyme gene is a gene selected from eucaryotic or procaryotic cells.

8. The method as claimed in claim 7, wherein the transferred gene is a gene which expresses nitroreductase, reductase, DT diaphorase, xanthine oxidase or other enzyme with reductive function.

9. The method as claimed in claim 5, wherein the labelled substrate is a compound with a nitro or another bioreducible structural component or a pharmaceutically acceptable salt thereof.

10. The method as claimed in claim 5, wherein the label is selected from the group consisting of 1231, 1241, 1311, 75Br, 76Br, 18F, 19F, 31 P, 13C and 11B.