WO1998002577A1

WO1998002577A1 - Method to co-detect introduced genes and their products

Info

Publication number: WO1998002577A1
Application number: PCT/US1997/010998
Authority: WO
Inventors: Louis M. Kunkel; Emanuela Gussoni
Original assignee: The Children's Medical Center Corporation
Priority date: 1996-07-15
Filing date: 1997-07-15
Publication date: 1998-01-22
Also published as: AU3717797A

Abstract

Methods of detecting and simultaneously visualizing, in a host cell, the presence of a single copy of an exogenous nucleic acid, and a protein encoded by the exogenous nucleic acid, are disclosed.

Description

METHOD TO CO-DETECT INTRODUCED GENES AND THEIR PRODUCTS

RELATED APPLICATIONS

This application claims priority to pending U.S. Patent Application No. 08/680,494, filed July 15, 1996, the content of which is incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with Government support under Contract Number NS23740 awarded by the National Institutes of Health. The Government has certain rights in the invention .

BACKGROUND OF THE INVENTION

Over the last decade, rapid progress has been made in the characterization and understanding of human genetic disorders leading to improved diagnostic tools and new insight into the molecular pathogenesis of genetic diseases (Collins, F.S., Nat . Genet., 5:347-350 (1995). The development of therapeutic strategies to correct or ameliorate primary genetic defects relies on replacement of the mutant gene with a wild-type DΝA copy that can be processed and expressed as the endogenous (natural) gene. Somatic gene therapy trials have been attempted in humans with replication-defective retroviruses and adenoviruses containing up to 8 kilobases of exogenous human DΝA for the treatment of familial hypercholesterolemia (Grossman, M. et al . , Na t . Genet . , 5:325-326 (1994); DeMatteo, R.P. et al . , Ann. Surg. , 222 : 229 - 239 (1995)), adenosine deaminase deficiency (ADA) (Bordignon, C. et al . , Science, 270:470- 475 (1995); Blaese, M . R . et al . , Science, 270:475-480 (1995)), and cystic fibrosis (Knowles, M.R. et al . , N. Engl . J. Med . , 333:823-831 (1995)).

Cell transfer techniques have also been used in human clinical trials to deliver wild-type genes or proteins to affected tissues for the treatment of genetic and neurodegenerative disorders. Allogenic fetal neural cells have been transplanted to the brain of patients with Parkinson's disease with some clinical improvements. Up to four years after transplantation, the donor cells survive and secrete dopamine (Freed, C. et al., N. Engl . J. Med . , 327:1549-1555 (1992); Widner, H. et al . , N. Engl . J. Med . , 327:1556-1563 (1992); Kordower, J. et al . , N. Engl . J. Med . , 332:1118-1124 (1995)). Similarly, human allogenic myoblasts have been transplanted into the muscles of patients affected by Duchenne muscular dystrophy (DMD) , in an attempt to deliver a normal copy of the DMD gene and allow expression of its encoded protein, dystrophin (Gussoni, E. et al , Na ture 356:435-438 (1992) ; Huard, J. et al . , Muscle & Nerve, 15:550-560 (1992); Karpati, G. et al . , Ann. Neurol . , 34:8-17 (1993); and Mendell, J.R. et al . , N. Engl . J. Med . , 333:832-838 (1995)).

In most human gene therapy clinical trials, the presence of input vectors or cells has been monitored by using the polymerase chain reaction (PCR) (Bordignon, C. et al , Science, 270:470-475 (1995); Blaese, M.R. et al . ,

Science, 270:475-480 (1995); Knowles, M.R. et al . , N. Engl . J. Med . , 333:823-831 (1995); and Gussoni, E. et al . , Nature 356:435-438 (1992)). However, the PCR techniques cannot provide answers to some of the most critical questions asked regarding the evaluation of gene transfer therapies, such as: 1) Where is the introduced DΝA within the target cell or tissue?; 2) How many copies of the introduced DΝA sequence are present in the target cells/tissue?; 3) Where is it located within the cell, in the nucleus or cytoplasm?; or 4 ) Is the desired protein expressed? Immunohistochemical detection can provide information on the intracellular location of an expressed protein (Wolff, J.A. et al . , Science, 247:1465-1468 (1990); Dhawan, J. et al . , Science 254:1509-1512 (1991); Lynch, C . et al . , Proc . Na tl . Acad . Sci . USA, 89:1138-1142 (1992); Stratford-Perricaudet , L.D. et al . , J. Clin . Invest . , 90:626-630 (1992); Acsadi , G. et al . , Hum. Molec . Genet . , 3:579-584 (1994)), but yields little information when no protein expression is detected. /Absence of the vector- encoded protein may be incorrectly interpreted as lack of infection or loss of vector DNA, when it may be the result of a failure of transcription or translation of the exogenous gene .

Techniques are also available to detect repetitive copies of chromosomal DNA (e.g., Speel, J. M. , et al . ,

His tochem . J. , 27:833-858 (1995) or multiple copies of mRNA and their encoded proteins (e.g., Speel, J. M., et al . , His tochem . J. , 27:833-858 (1995); Egger et al . , J. His tochem . Cytoche . , 42:815-822 (1994); Gingras , D., et al . , Cell Vision , 2:218-225 (1995)). However, none of these techniques are sensitive enough to measure a single copy of DNA, with the simultaneous co-detection of the protein encoded by the introduced image-copy DNA molecule which is a critical determination to evaluate a gene therapy protocol.

There exists a great need for an efficient and cost- effective method of evaluating gene therapy studies that is sensitive enough to measure single copies of introduced genes, or partial gene sequences, and which provides information regarding the microenvironment in which the delivered genes reside and the effects of that environment on gene expression. SUMMARY OF THE INVENTION

The present invention relates to methods of simultaneously detecting, in a target cell or tissue sample, the presence of an exogenous nucleic acid and a protein encoded by that exogenous nucleic acid. As used herein, the term "exogenous" means that the nucleic acid is introduced from outside of the target cell or tissue, that is, that the nucleic acid is not produced or synthesized within the cell or tissue. The term "introduced" is also used herein to describe the exogenous nucleic acid. The exogenous nucleic acid can be any DNA or RNA that encodes a protein, e.g., a gene, or partial gene sequence, or a virus. Specifically encompassed by the present invention are methods to simultaneously detect a single copy of a gene introduced into target cells and the expression of the protein encoded by the introduced gene.

The methods described herein comprise immunohistochemistry with an antibody that binds specifically to the protein encoded by the exogenous nucleic acid, and in-situ hybridization of a nucleic acid probe that hybridizes specifically to the exogenous nucleic acid of interest, and the simultaneous detection and visualization of the nucleic acid molecule and its expressed protein product . The methods of the present invention can be used to detect the exogenous nucleic acid and its encoded protein in any biological sample that contains cells. Biological samples specifically encompassed by the present invention include tissue samples, such as, e.g., skeletal muscle, brain, skin and internal organs such as kidney or liver. Typically, a tissue sample which has been sectioned appropriately for immunohistochemistry and in-situ hybridization, is obtained that contains cells into which the exogenous DNA has been introduced. The sample is contacted with an antibody that is specific for the protein encoded by the introduced nucleic acid of interest, under conditions which allow specific binding of the antibody to the protein. Subsequently, the sample is contacted with the nucleic acid probe, under conditions which allow specific hybridization of the nucleic acid probe to the exogenous DNA. The presence (or absence) of binding of the antibody to the peptide, as well as the presence (or absence) of hybridization of the nucleic acid probe to the exogenous nucleic acid of interest, are then simultaneously visualized in a microscope field using a fluorescence microscope. Binding of the antibody to its target protein is indicative of the presence of the protein in cells of the sample, e.g., tissue sections, and hybridization of the nucleic acid probe to the introduced nucleic acid is indicative of the presence of the introduced gene in cells of the sample (e.g., tissue sections).

To facilitate visualization of binding of the antibody to the protein encoded by the exogenous introduced DNA, as well as hybridization of the nucleic acid probe to the introduced DNA of interest, both the antibody to the peptide, and the nucleic acid probe can be labelled. Appropriate labels include fluorophores, which can be conjugated to either the probe or the antibody; reporter molecules, such as nucleotide analogs (e.g., biotin, digoxigenin, or sulfhydryl analogs) , which are recognized by reporter-binding molecules, such as avidin, streptavidin, anti-digoxigenin antibody, or other reporter- binding molecules; and other appropriate labels. In one embodiment, the antibody to the protein is detected through the use of a labelled secondary antibody that binds to the antibody to the protein, and the nucleic acid probe is labelled with digoxigenin. Hybridization of the probe with the exogenous DNA, e.g., the single copy DNA molecule, is detected through the use of a labelled anti-digoxigenin antibody. Most typically, the anti-digoxigenin antibody and the secondary antibody are labelled with fluorophores. The labels are selected to allow the co-detection and simultaneous visualization of the presence (or absence) of binding of the antibody to the protein, as well as the presence (or absence) of hybridization of the nucleic acid probe to the introduced nucleic acid, for the evaluation of gene therapy experiments, including human clinical trials,

Because of the work described herein, methods are now available, for the first time, that allow efficient detection of a single copy of a nucleic acid molecule (the exogenous nucleic acid of interest) , in a cell/tissue sample, as well as co-detection and simultaneous visualization of the encoded protein. The methods permit visualization of the environment in which the introduced nucleic acid of interest is located as well as determination of the copy number of the introduced nucleic acid of interest in the recipient cells, and the expression of the protein. The methods described here can be used for a broad range of exogenous nucleic acids of interest and proteins. The methods provide for the first time a means to assess the efficacy of gene therapy by co-detection and visualization of the presence and expression of an exogenous nucleic acid within the cellular environment .

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods of co- detecting and simultaneously visualizing single copies of DNA and determining the presence or absence of its protein product in tissue samples. The methods described herein comprise immunohistochemical detection and in-situ hybridization and provide an efficient and cost-e fective means to evaluate gene therapy protocols by specifically following the fate of an introduced nucleic acid of interest and a protein or protein encoded by the nucleic acid. The methods of the present invention are significantly more sensitive than currently available methods and can detect single copies of a nucleic acid molecule, or even a partial gene sequence, that are present in a cell or tissue sample. Because of this sensitivity, results of these methods provide a great deal of information useful to evaluate gene therapy protocols . For example, information regarding the number of copies of DNA present in tissue after gene, or cell injection in gene therapy protocols, and the location within the cell or tissue of the introduced DNA and its encoded protein (e.g., is the introduced gene present within the cell, and specifically within the nucleus?) can be determined using the methods described herein.

As described below in the Examples, exogenous gene localization and expression was investigated using two therapeutic protocols. In the first protocol, a plasmid herpes simplex virus vector (pHSVlac) expressing beta- galactosidase {jβ-Gal) was used to deliver β-Gal to the brain of rodents. Vector DNA was localized in the cell nuclei of brain tissue samples; however, not all of the nuclei containing the vector DNA produced S-Gal, suggesting that some step in either transcription or translation of the exogenous gene was compromised. Furthermore, nuclei were observed with more than one hybridization signal in some _/S-Gal positive cells. Since the introduced vectors were replication-defective, this suggested that the same cell may have been infected by multiple vector particles. In the second protocol, normal human myoblasts were injected into muscle to deliver the dystrophin gene and protein in DMD patients participating in a phase-one therapeutic trial. Through examination of muscle samples, it was possible to detect donor nuclei that both did and did not express dystrophin. In some cases, donor nuclei were seen surrounded by host nuclei. As a result of these experiments, methods are now available to detect concurrently in a host cell or tissues single copies of an exogenous nucleic acid of interest that has been introduced into the host cell, as well as a protein of interest. As referred to herein, an "exogenous nucleic acid of interest" or "introduced nucleic acid of interest" is a nucleic acid containing a polynucleotide encoding a protein, which is not found naturally in the host cells, or is not expressed because of a DNA mutation. Typically, the exogenous nucleic acid is a gene suitable for use in a gene therapy protocol. The exogenous gene is typically inserted into a vector suitable to introduce the gene into a target cell, and therefore can also include other elements, such as promoter elements, enhancer elements, splicing signals, termination and polyadenylation signals, viral replicons, bacterial plasmid sequences, or other vector nucleic acid sequences. The "encoded protein", also referred to as the "protein of interest," is typically the protein that is encoded by the polynucleotide, e.g., the protein product of the gene.

Alternatively, a protein of interest is not encoded by the exogenous gene, but is of value to determine the environment surrounding the introduced gene, e.g., a cell surface protein such as CD3 , to determine whether an immune response has been initiated by the host to the introduced gene/cells .

"Host cells" or "target cells" are any cells, including cells of a tissue, into which an exogenous nucleic acid of interest has been introduced. In a preferred embodiment, the host cells are mammalian cells, particularly human cells. The host cells can be part of a tissue, or individual cells.

The exogenous DNA of interest can be introduced to the host cells by a wide variety of methods. For example, the exogenous DNA can be introduced in a vector, or via a genetically altered virus containing the exogenous nucleic acid of interest. Alternatively, the exogenous DNA that is either absent from, or defectively produced in a target cell (the "recipient cell"), can be delivered via normal donor cells. For example, in Duchenne 's Muscular Dystrophy (DMD) , a portion of the DMD gene may be deleted or mutated, resulting in the absence of expression of its encoded protein, dystrophin. Normal donor myoblasts containing the complete DMD gene are introduced into the muscle of DMD patients. Normal donor cells may fuse with recipient muscle fibers and, as a result, dystrophin may be expressed. Alternatively, the exogenous DNA of interest can also be introduced into a donor cell, which is then delivered to the recipient tissue (s) . Prior to performing the methods of the invention, a sample of the host cells to be examined for the presence of the exogenous nucleic acid of interest and the protein of interest (the "test sample") is obtained. The test sample can comprise individual cells, e.g., cultured cells, or, typically a tissue sample, e.g., a blood or biopsy sample. The test sample is preserved and sectioned using standard methods, to prepare the sample for in-situ hybridization and immunohistochemistry (see Ausubel, F.M. et al . , eds . , Current Protocols in Molecular Biology, John Wiley & Sons, 1994) . For example, the test sample can be cryogenically preserved. If the test sample consists of a tissue sample, the tissues can be perfused during preparation. The test sample can be fixed, using an appropriate fixative. Fixation should not proceed to the point at which antigenic activity of proteins in the sample is lost. The test samples are also sectioned, using appropriate methods.

Two parameters to be optimized during preparation of the test sample include the method of cell or tissue fixation, and the section thickness of the tissue. For example, with regard to the fixation methods described in the Examples below, very few hybridization signals were seen during in-situ hybridization when formalin was used as a fixative, possibly due to restricted probe access in extensively cross-linked formalin- fixed tissue. When a non cross- linking fixative (Histochoice™, Amresco, Solon, OH) was tested, positive hybridizing signals were seen reproducibly in different types of tissue. Fixation was also found to influence protein detection by immunohistochemistry. For example, dystrophin detection was more sensitive when tissue sections were fixed in ethanol rather than Histochoice™. Optimization of fixation methods for different exogenous nucleic acids of interest and their encoded proteins can be performed using routine experimentation and standard methods (see, for example, Ausubel, F.M. et al . , eds . , Current Protocols in Molecular Biology, John Wiley & Sons, 1994) . In a preferred embodiment, the whole sample is cryogenically preserved and sectioned, and then fixed in an appropriate fixative, such as methanol, before immunohistochemistry is performed, in order to maintain sensitivity of the sample during immunohistochemistry.

Tissue section thickness is also an important parameter for hybridization efficiency during in-situ hybridization. Cell nuclei in muscle and brain tissues have an average size of 15-20 μm (Landon, D.N. , Skeletal muscl e pa thology, 1:1-87, F.L. Mastaglia and S.J. Walton (eds.) (1982); Zagon, I.S. and McLaughin, J. Brain Res . , 170:443-457 (1979); Smialowska, M. et al . , Neurosci . , 26 : 803 - 801 (1988), larger than the thickness of the tissue sections, which results in a decreased number of expected hybridization signals, since many nuclei are themselves sectioned and may therefore lack the DNA sequence of interest. Thus, tissue section thickness should be taken into account when interpreting results. Section thickness is determined to maximize access of probes to the target DNA, as well as access of signal detection agents. Optimal tissue thickness can be determined using routine experimentation and standard methods (see, for example, Ausubel, F.M. et al . , eds., Current Protocols in Molecular Biology, John Wiley & Sons, 1994) .

Test samples which have been preserved and sectioned, as described above, are referred to herein as "prepared samples." The methods of the current invention are performed on the prepared samples. The methods include sequentially performing immunohistochemistry and in-situ hybridization, and then simultaneously visualizing the exogenous nucleic acid of interest and the encoded protein. "Immunohistochemistry" refers to the detection of the protein of interest through the specific binding of an antibody with the protein. "Specific binding" of an antibody indicates that the antibody binds solely to the protein, and not to other proteins that are present in the prepared sample. The antibody that binds specifically to the protein of interest is also referred to herein as the "primary antibody" .

The term "antibody", as used herein, encompasses both polyclonal and monoclonal antibodies, as well as mixtures of more than one antibody reactive with the protein of interest (e.g., a cocktail of different types of monoclonal antibodies reactive with the protein) . The term antibody is further intended to encompass whole antibodies and/or biologically functional fragments thereof, chimeric antibodies comprising portions from more than one species, humanized antibodies, human- like antibodies, and bifunctional antibodies. Biologically functional antibody fragments which can be used are those fragments sufficient for binding of the antibody fragment to the protein of interest. The chimeric antibodies can comprise portions derived from two different species (e.g., a constant region from one species and variable or binding regions from another species) . Antibodies can be produced using routine experimentation and standard methods (see, for example, Current Protocols in Immunology, John Wiley & Sons, 1995) . The antibody used in immunohistochemistry can be labelled to facilitate detection. Representative labels include fluorescent labels, such as fluorescein isothiocyanate (FITC) , tetramethyl rhodamine isothiocyanate (TRITC) , Texas red, phycoerythrin, or other fluorochrome . The antibody can be labelled with a directly detectable label, such as by conjugation of the antibody to a fluorochrome . Preferably, it can be labelled with an indirectly detectable label in order to enhance the detectable signal. Representative indirectly detectable labels include antibodies that are bound by a secondary antibody that recognizes and is specific to the primary antibody; or PAP-immunoperoxidase labelling, where the primary antibody is coupled to PAP complex by a bridging antibody. If a secondary antibody is used, it can be labelled, such as by a fluorophore; alternatively, the secondary antibody can be biotinylated, and can be detected by interaction with a streptavidin fluorochrome . It should be noted that although the primary antibody specifically binds to the encoded protein, "background" nonspecific binding of the secondary antibody to other components of the prepared sample may also occur. The type of label will vary, depending on the antibody, the protein, the method of detection used, the results sought, and other factors. In a preferred embodiment, the primary antibody is detected with a secondary, anti-IgG antibody conjugated with FITC. To perform immunohistochemistry, the prepared sample is contacted with the primary antibody, under conditions so that specific binding of the primary antibody to the protein of interest, if it is present in the prepared sample, can occur. If the primary antibody is detected by binding of a secondary antibody to the primary antibody, the prepared sample is contacted with the primary antibody and the secondary antibody, under conditions so that specific binding of the primary antibody to the protein, as well as binding of the secondary antibody to the primary antibody, occurs. Following immunohistochemistry, the prepared sample is fixed, using an appropriate fixative, such as Histochoice™, as described above. This fixation step will preserve the protein staining even after in-situ hybridization is performed. The prepared sample can also be counterstained to highlight cell structures, such as cell nuclei. Counterstaining allows visualization of the exact position of the exogenous nucleic acid of interest and the encoded protein. For example, counterstaining with 4' -6'- diamidino-2-phenylindole (DAPI) facilitates determination of whether the nucleic acid of interest is within the nucleus of the host cell. Similarly, counterstaining will facilitate determination of whether a protein of interest has been exported out of the host cell. After immunohistochemistry, in-situ hybridization is performed. The term, "in-situ hybridization", as used herein, refers to the hybridization of a nucleic acid probe to the exogenous single copy DNA molecules introduced via a vector or donor cells, in the prepared sample. Hybridization should be performed under stringency conditions that allow specific hybridization of the nucleic acid probe to the exogenous DNA. "Specific hybridization" indicates that the nucleic acid probe hybridizes solely to the exogenous DNA, and not to other genes or nucleic acids in the prepared sample. It should be noted that although the probe specifically hybridizes to the exogenous nucleic acid of interest, "background" nonspecific hybridization of the probe to other components of the prepared sample may also occur. Hybridization conditions will vary, depending on the probe used, the exogenous nucleic acid of interest, the species of the host, and other factors. Such specific conditions of hybridization are known to one of skill in the art, or can be determined empirically using standard laboratory techniques. Representative hybridization conditions that will allow specific hybridization can be found in Current Protocols in Molecular Biology (Ausubel, F.M. et al . , eds., John Wiley & Sons, 1994).

The probe used for in-situ hybridization is a nucleic acid that specifically hybridizes to a single copy of a target DNA. The probe can hybridize to the polynucleotide that encodes the protein of interest; alternatively or in addition, the probe can hybridize to other sequences in the exogenous nucleic acid (e.g., vector sequences, promoter or enhancer elements, etc.). The probe can be generated using standard methods (see Ausubel, F.M. et al . , eds., Current Protocols in Molecular Biology, John Wiley & Sons, 1994) . In a preferred embodiment, the probes are generated by fragmentation of the nucleic acid of interest: the fragments of the single-copy nucleic acid of interest are used as the nucleic acid probes. Probes generally range from approximately 50-500 base pairs (bp) in final length, although larger or smaller probes can be used, depending on the level of efficiency of hybridization that is sought. For example, in the Examples described below, probes of a final size of 150-200 bp hybridized most efficiently and gave the least background to tissue sections, whereas shorter probes (75-150 bp) gave less efficient detection of hybridization signals. Probes of 450-500 bp in size generated high background due to nonspecific binding to the tissue sections. Smaller probe size facilitates entry of the probe into the cell. Optimal probe size can be determined using routine experimentation and standard methods (see, for example, Ausubel, F.M. et al . , eds., Current Protocols in Molecular Biology, John Wiley & Sons, 1994) . In a preferred embodiment, the probe is approximately 150-200 bp in length, because of efficient hybridization.

The probe used for in-situ hybridization can be labelled to facilitate detection. Representative labels include fluorescent labels, such as fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethyl rhodamine isothiocyanate (TRITC) , Texas red, phycoerythrin, or other luorochrome . The probe can be labelled with a directly detectable label, such as by attachment of a fluorochrome to the probe. Alternatively, it can be labelled with an indirectly detectable label, such as by conjugation of the probe with a reporter molecule (e.g., biotin, digoxigenin) , which can be recognized by a reporter-binding molecule (e.g., avidin, streptavidin, or digoxigenin antibody) . The reporter-binding molecule can be attached to a fluorescent label, or to an enzyme (e.g., alkaline phosphatase or horseradish peroxidase) for enzymatic detection. The type of label will vary, depending on the size of the probe, the method of detection used, the results sought, and other factors. In a preferred embodiment, the probe is labelled with a reporter molecule, such as digoxigenin, which is recognized by a reporter-binding molecule, such as an anti-digoxigenin antibody, attached to a fluorophore. This preferred embodiment amplifies the signal, facilitating detection.

After protein detection and post-fixation, to conduct in-situ hybridization, the prepared samples are denatured, using standard methods, and are contacted with the probe, under conditions for specific hybridization of the probe to the exogenous DNA to occur (if the exogenous nucleic acid of interest is present) . If the probe is labelled with an indirectly detectable label (e.g., a reporter molecule, such as digoxigenin) , the prepared samples are contacted with the probe containing the reporter-binding molecule . The conditions are tailored to allow specific hybridization of the probe to the exogenous nucleic acid of interest, as well as binding of the reporter-binding molecule to the reporter molecule.

It should be noted that the characteristics of the exogenous nucleic acid of interest may affect in-situ hybridization. For example, fluorescent in-situ hybridization (FISH) is highly efficient at detecting unique target sequences of genomic DNA of 12-30 kb in size, but the hybridization efficiency decreases when the target sequence is smaller than 2 kb (Trask, B.J., Trends Genet . , 7:149-154 (1991). In the gene therapy setting, it is critical to detect unique, single copy sequences together with the protein they encode. Appropriate alterations in the in-situ hybridization procedures can be determined using routine experimentation and standard methods (see, for example, Ausubel, F.M. et al . , eds., Current Protocols in Molecular Biology, John Wiley & Sons, 1994) . Typically, the target sequence is a nucleic acid of interest of approximately 5-40 kb in size. After in-situ hybridization and immunohistochemistry steps are performed, as described above, the presence of hybridization of the probe to the exogenous nucleic acid of interest, and the presence of binding of the antibody to the encoded protein, are detected (visualized) simultaneously. The appropriate detection method depends on the type of labelling of the probe and antibody. For example, if fluorescent labels are used for both the probe and the antibody, the presence of hybridization of the probe and binding of the antibody can be visualized simultaneously by fluorescence microscopy. If antibody and the probe are both fluorescently labelled, fluorochromes which emit light of different detectable wavelengths (e.g., visualized as different colors) should be used for the probe and the antibody. In a preferred embodiment, the primary antibody recognizing the protein of interest is labelled with FITC, or with a primary antibody that is recognized by a secondary antibody which is conjugated to FITC; and the probe is labelled with digoxigenin. The presence of digoxigenin is detected with fluorescently- labelled anti-digoxigenin antibody.

Detection of hybridization of the probe to the introduced single copy DNA molecule indicates whether the DNA present in the prepared sample. Interaction between the primary antibody and the protein of interest indicates whether the protein of interest is present as well.

The methods described here are highly efficient at detection of a single nucleic acid molecule, and simultaneous visualization of the protein encoded by that DNA. The methods add a new dimension to gene therapy studies by visualizing the environment in which the introduced nucleic acid of interest or cells are located, something not possible when reverse- transcribed RNA or DNA extracted from tissues are analyzed using PCR.

The co-detection of exogenous DNA using in-situ hybridization combined with immunohistochemistry allows the determination of the copy number of the introduced nucleic acid of interest in the recipient cells and the expression of the encoded protein over time.

Alternatively, immunocytochemistry with an antibody that does not recognize the protein encoded by the introduced gene, but recognizes, e.g., a host protein, can be used to detect a protein that provides information regarding the presence of an immune reaction from the host to the introduced gene/cells. The methods described here are sensitive and powerful tools that can be applied to a broad range of therapeutic studies . Regardless of the type of biological vector used in the experiment, it is possible to design probes that specifically hybridize to unique DNA sequences of the introduced nucleic acid of interests and to co-detect the protein products, thus providing a very sensitive way of following the long-term fate of input genes, and evaluating the success of gene therapy.

The invention is further described by the following Examples.

EXAMPLE 1 : MATERIALS AND METHODS

Rodent Samples . Three male Sprague Dawley rats (250g weight) were stereotactically injected in the striatum on one side of the brain with 1.8 x IO⁴ infectious particles (IVP) of pHSVlac and mock injected with 10% sucrose on the contralateral side as described (During, M.J. et al . , Sci ence, 266 : 1399 - 14.02 (1994)). Helper virus-free vector particles containing multiple copies of the plas id pHSVlac (Geller, A.I. and Breakefield, X.O., Science, 241:1667 -1669 (1988); Geller, A.I. and Freese, A., Proc . Na tl . Acad . Sci . USA, 87:1149-1153 (1990); Geller, A.I. et al . , Proc . Na tl . Acad . Sci . USA, 90:7603-7607 (1993)) were prepared as described (Fraefel, C, J. Virol, in press (1996)) . A fourth animal was mock-injected on both sides of the brain. Animals were sacrificed 7 days after the date of injection. Brains were isolated, rapidly frozen in chilled isopentane and stored at -70°C.

Human Samples . Peripheral blood lymphocytes were isolated from a normal emale. Cells were cultured and harvested as described for the preparation of slides for cytogenic analysis (Verma, R.S. and Babu, A. (eds) , Human Chromosomes - Manual of Basic Techniques (Pergamon Press, New York (1989) ) . Normal human skeletal muscle was obtained from a 29 year old male during a surgical procedure. Tissue was snap-frozen in liquid nitrogen and stored at -70°C.

Bilateral biopsies were taken from the tibialis anteriors (TA) before transplantation, as well as 1 month and 6 months after myoblast transfer from DMD patients who participated in a myoblast transfer clinical trial (Gussoni, E. et al . , Nature, 356 : 435-438 (1992)). All patients had a deletion in the dystrophin gene, as assayed by multiplex PCR (Beggs, A.H. et al . , Hum . Genet . , 86:45- 48 (1990)) . One TA muscle of each DMD patient was injected with myoblasts from a normal donor, while the contralateral TA was mock-injected (Gussoni, E. et al . , Na ture, 356:435- 438 (1992)). Tissue was rapidly frozen in freezing isopentane and stored at -70°C. Immunohistochemistry. 10 μm sections were collected from the injected rat brains at -22°C and fixed for 45 min at room temperature using Histochoice™ (Amresco, Solon, OH), a non-crosslinking fixative, according to the manufacturer's instructions. For 0-Gal detection, sections were blocked for 20 minutes at room temperature in PBS and 10% fetal calf serum, and then incubated for 14 hours at 4°C with a 1:100 dilution of an anti-β-Gal antibody (Sigma Immunochemicals, St. Louis, MO). Washes were performed in ice-cold PBS three times for 10 minutes each. Sections were then incubated for 90 minutes at room temperature with an anti-mouse IgG antibody conjugated with FITC (1:50; Boehringer Mannheim, Indianapolis, IN), washed in PBS as above, mounted in Vectashield (Vector Labs, Burlingame, CA) and examined using a Zeiss Axiophot microscope . Muscle biopsies were sectioned at -20°C in 10 μm sections. Sections were fixed in methanol for 3 minutes at room temperature, transferred to ice-cold PBS for 5 minutes, and blocked as described above. Dystrophin was detected by incubating the sections for 14 hours at 4°C with an affinity-purified polyclonal antibody which recognizes the dystrophin distal "rod domain' (cDNA residues 6,181-9,544) (0.3 μg/ml) (Lidov, H.G.W. et al . , Na ture, 348:725-728 (1990); Byers , T.J. et al . , J. Cel l Biol . , 115:411-421 (1991)). After washing in ice-cold PBS as above, sections were incubated with an anti-rabbit IgG antibody conjugated with FITC (1:50; Boehringer Mannheim, Indianapolis, IN) and washed as described for brain tissue sections .

In -si tu hybridiza tion . Following immunohistochemistry, for the simultaneous detection of both the exogenous gene and its encoded protein, care was taken to prevent exposure of slides to the light, and all of the following incubations were performed in Coplin jars covered with aluminum foil. Slides were incubated in PBS for 10 minutes at room temperature, and coverslips were removed using a 26¹² gauge needle. Brain or muscle tissue sections were refixed for 45 minutes in Histochoice™ (Amresco, Solon, OH) according to the manufacturer's instructions and stored in a humidified chamber at 45°C until denaturation.

Probes . Genomic rat probe DH1 contains a sequence of approximately 10 kb of the rat DH1 single -copy gene, encoding for the enzyme core 2 GlcNAc-T (Nishio, Y. et al . , J. Cl in . Invest . , 96:1759-1767 (1995)). pHSVlac is a 8 kb plasmid vector previously described (Geller, A.I. and Freese, A., Proc . Na tl . Acad . Sci . USA, 87:1149-1153 (1990) ) .

Overlapping genomic phages containing dystrophin exons 10, 11 and 12 (W4 , W5 , W6 ) (Monaco, A. P. et al . , Na ture , 323:646-650 (1986)) with an average insert size of 13.5 kb were pooled and tested as a probe (dys 10-12) . Similarly, a genomic phage λ IB 48 containing a 12.8 kb sequence flanking and including dystrophin exon 48 was used as a probe (dys 48) . For the preparation of each probe, one microgram of phage or plasmid DNA was incubated at 37°C for 30 minutes in the presence of 12.5 μg/ l RNase A. Probe DNA was labeled with digoxigenin-11 -dUTP by nick translation as previously described (Lichter, P. et al., Proc . Na tl . Acad . Sci . USA , 85:9664-9668 (1988); Lichter, P. et al . , Sci ence, 247:64-69 (1989)). Samples were incubated at 16°C for 60- 90 minutes to a final product size ranging from 200-400 bp. The size of labeled probes was checked by electrophoresis of 1/10 reaction volume on a 2% agarose gel. Probes dys 10-12 and dys 48 were precipitated with 100 μg Cot I digested human DNA (Boehringer Mannheim) while DH1 and pHSVlac were precipitated with rat genomic DNA digested to 200 bp size. DNA was pelleted by centrifugation at 14,000 rpm at 4°C, rinsed with cold 70% ethanol, dried and resuspended in 50μl IX hybridization cocktail (50% deionized formamide, IX SSC (20X SSC: 3M NaCl, 0.3M Na₃citrate2H₂0, pH 7.0), 10% dextran sulfate, IX Denhardt ' s (100X Denhardt' s: 2% bovine serum albumin, 2% Ficoll, 2% polyvinylpyrrolidone) . In -si tu hybridization . Sections were denatured for 12 minutes in 70% formamide (American Bioanalytical , Natick, MA) 2X SSC pre-warmed to 70°C and immediately dehydrated in pre-chilled ethanol series (50%, 70%, 90%, 100%) . Probes were also denatured at 70°C for 20 minutes and transferred to a 37 °C waterbath for 10 minutes.

Ten μl of probe (200 ng) were added to each slide, sections were covered with an 18 x 18 mm coverslip, sealed with rubber cement and incubated for 15 hours at 45 °C in a humidified chamber. Sections were washed 3 times for 5 minutes in 50% formamide 2X SSC pre-warmed to 45°C, followed by 3 times for 5 minutes in 0. IX SSC pre-warmed to 60°C. Slides were incubated in a humidified chamber at 37°C for 30 minutes with digoxigenin blocking solution (150 mM NaCl, 100 mM Tris pH 7.5, 0.5% non-fat dry milk), then for an additional 30 minutes with an anti-digoxigenin antibody conjugated with rhodamine (Boehringer Mannheim, Indianapolis, IN) diluted in digoxigenin blocking solution (2 μg/ml) . Sections were washed 3 times for 5 minutes in 150 M NaCl, 100 mM Tris pH 7.5 , 0.5% Tween-20 pre-warmed to 45°C. Nuclei were counterstained with 4' -6' diamidino-2-phenylindole (DAPI) (200 ng/ml) diluted in Vectashield mounting medium. Slides were examined using a Zeiss Axiophot microscope.

Images . After immunohistochemistry and FISH were performed on same tissue sections, visual inspection of the results through a triple band-pass filter that allows the simultaneous visualization of multiple colors (Omega, Brattleboro, VT) revealed the preservation of the protein signal by immunohistochemistry and the simultaneous detection of the DNA hybridization signal over the DAPI counterstained nuclei. Images were collected from the same microscopic field with separate filters for DAPI, FITC and rhodamine signals using a CCD camera (Photometries, Tucson, AZ) . The images were directly overlaid on one another using a Macintosh computer and IPLab Spectrum SU2 software (Signal Analytics, Vienna, VA) . Photographs from different microscopic fields were further gathered in figure panels using Adobe Photoshop and ClarisDraw softwares.

EXAMPLE 2 : FISH OPTIMIZATION In an effort to optimize the detection of introduced genes by FISH, hybridization probes were tested on three normal tissue types: human peripheral blood lymphocytes, human skeletal muscle and rat brain tissue sections. Hybridization probes used included dys 10-12, which recognizes dystrophin exons 10-12, and probe dys 48, which recognizes the sequences surrounding and including dystrophin exon 48. The dystrophin probes were first independently tested for hybridization efficiency on nuclei derived from peripheral blood lymphocytes of a normal female. Under these experimental conditions, the efficiency of probe dys 10-12 was 96%, while for probe dys 48 was 94% (Table 1) . Hybridization efficiency was expressed as the ratio between the number of positive signals seen in a given number of nuclei over the total number of positive signals expected (Table 1) . Next, hybridization to normal male skeletal muscle tissue sections with these probes was evaluated in five different experiments (Table 1) . The highest hybridization efficiency of probe dys 10-12 was 60%, as tested in three separate experiments, whereas the efficiency of probe dys 48 was 67% (Table 1) . Variations in hybridization efficiency could be attributed to the end size of the probe, with approximately 150-200 bp being the most efficient size (Table 1) . The observed difference in hybridization efficiency between lymphocytes and muscle tissue sections can in part be attributed to the average size of cell nuclei in human muscle being larger than the section thickness (Landon, D.N., pp. 1-87 in Skeletal muscle pathology (F.L. Mastaglia and S.J. Walton, eds., Churchill Livingstone, London, 1982)), thus on any given section, part of the nucleus is missing. In contrast, with lymphocytes, whole cell nuclei were available for hybridization.

Table 1. Hybridization efficiency of FISH probes tested on normal tissues

Probe Tissue Nuclei¹ Expected² Positive³ Efficiency⁴ Probe size (bp)

Dys 10-12 Lympho100 200 193 96% 150-200 cytes

Dys 10-12 exp.l Muscle 203 203 121 60% 150-200

Dys 10-12 exp.2 Muscle 200 200 110 55% 200-250

Dys 10-12 exp.3 Muscle 300 300 123 41% 70-150

Dys 48 Lympho100 200 189 94% 150-200 cytes

Dys 48 Muscle 100 100 67 67% 150-200

Dys 48 Muscle 200 200 109 54% 100-200

DH1 exp.l Brain 200 400 173 43% 250-300

DH1 exp.2 Brain 200 400 167 41% 300-400

DH1 exp-3 Brain 200 400 156 39% 350-450

Total nuclei counted

Expected number of positive signals

Observed number of positive signals

The proportion of hybridization signals detected versus those expected was calculated as the percentage of the ratio between the number of positive hybridizing signals counted¹³¹ over the total expected ^l2) .

The efficiency of FISH was additionally tested on rat brain tissue sections using probe DHl recognizing a single copy gene in the rat genome. In one nucleus there were two hybridization signs, whereas others had only one. The highest efficiency of probe DHl tested in three separate experiments was 43% (Table 1) . Probe efficiency was calculated as above, taking into account that probe DHl was expected to recognize two sites in each nucleus. As for human muscle, the size of cell nuclei in rat brain is larger than the section thickness, thus partially explaining the lowered hybridization efficiency (Zagon, I.S. and McLaughin, J. Brain Res . , 170:443-457 (1979); Smialowska, M. et al . , Neurosci . , 26 : 803 - 801 (1988)) .

EXAMPLE 3 : HSV DETECTION IN RAT BRAIN To apply this technique to animals undergoing an experimental gene therapy protocol, tissue sections were collected from three rats injected in one side of the brain with a pHSVlac vector and placebo in the contralateral side, and from a fourth animal injected with placebo in both sides. Brain sections were hybridized using pHSVlac

DNA as hybridization probe. The pHSVlac vector is packaged in replication-defective HSV-1 particles as a DNA concatenate (Geller, A.I. and Breakefield, X.O., Sci ence , 241:1667-1669 (1988); Geller, A.I. and Freese, A., Proc . Na tl . Acad . Sci . USA, -97:1149-1153 (1990); Geller, A.I. et al . , Proc . Na tl . Acad . Sci . USA , 87:8950-8954 (1990)), making its detection easier than in the control case of DHl (single copy gene) . Nuclei in each section were stained in blue with DAPI, while hybridization signals were shown by red dots. Viral particles were detected on brain sections as positively hybridizing signals in the vector- injected side. Three areas of the virus-injected side of the brain demonstrated several positive hybridization signals. No positive signal was seen in the mock-injected side or in the animal solely injected with placebo. Vector sequences were seen within nuclei, as well as in areas surrounding cell nuclei. Some cell nuclei showed more than one hybridization signal.

Although viral particles had been detected by FISH in cell nuclei of vector-injected animals, it was unclear whether the reporter gene β-Gal was co-expressed in all vector-DNA positive cells or only in some. To co-localize protein and vector DNA, brain sections from all four animals were collected and stained by immunohistochemistry with an antibody recognizing the protein _/S-Gal. After immunohistochemistry, tissue sections were subsequently hybridized by FISH with probe pHSVlac.

Vector sequences were seen in cells co-expressing _/S-Gal. The protein /3-gal was visualized in green, the FISH hybridization signals were stained in red, and nuclei were in blue. In some 3-Gal positive cells, two distinct hybridization signals were detected, as shown by the presence of two red dots, suggesting that two distinct infections may have occurred. Positive hybridization signal was seen in both nuclei. The nucleus showed presence of vector DNA (red dot) accompanied by production of the encoded protein (green) . The other nucleus in this field also contained pHSVlac DNA sequences, as shown by the red dot, but no β-gal protein was detected, as shown by the absence of green staining. Many or more than half of the nuclei carrying the delivery vector pHSVlac were negative for S-Gal staining, implying the absence of protein expression in many of the infected cells. It was possible to detect simultaneously the vector DNA and the expressed protein because the protein staining remained after FISH was performed, and it was clearly visible. EXAMPLE 4 : DONOR CELL DETECTION IN MYOBLAST TRANSPLANT

In the second gene detection test, muscle tissue sections from a DMD patient participating in a myoblast transfer clinical trial were examined. This patient (patient #5) was previously reported as expressing donor dystrophin RNA as detected by PCR (Gussoni, E. et al., Na ture , 356:435-438 (1992) . The patient, deleted for dystrophin exon 48 and its surrounding DΝA, gave a unique opportunity to detect donor myoblasts by FISH using probe dys 48, since only donor-derived nuclei would exhibit a positive hybridization signal. Muscle tissue sections from biopsies taken before and after myoblast transfer were analyzed. Nuclei were stained in blue with DAPI, while positive hybridization signals were in red. Hybridizing donor nuclei (having a red dot) were found surrounded by mononuclear cells presumably from the host or nearby host myofibers. Positively hybridizing donor nuclei were detected in the myoblast-injected muscle biopsy of the patient, but not in the pre-implant nor in the placebo- injected ones. Occasionally, positively hybridizing donor nuclei were found surrounded by non-hybridizing nuclei. Donor nuclei were also seen together with negative nuclei in the proximity of a myofiber, suggesting that donor myoblasts may have fused to host myofibers. To determine whether the donor nuclei were within a myofiber membrane, the patient's muscle sections were stained using an anti- dystrophin antibody. As with the previous brain study, immunohistochemistry was followed by FISH on the same tissue sections to co-detect donor nuclei and confirm protein expression. The results of this combined experiment on a normal muscle tissue section and on a section from the myoblast-injected sample of the DMD patient . Dystrophin was visualized in green at the sarcolemma of the muscle fiber. The FISH hybridization was visualized in red. The sarcolemma of the muscle fiber was stained green for dystrophin, and the positive hybridization signal (red dot) was simultaneously localized in the nucleus. In the donor nucleus with a positive hybridization signal was surrounded by the green sarcolemmal staining of dystrophin, indicating that a donor myoblast had indeed fused with the pre-existing muscle fiber of the patient, resulting in dystrophin production.

EXAMPLE 5: CO-DETECTION OF INTRODUCED GENES/CELLS WITH THEIR ENCODED PROTEIN PRODUCT OR WITH OTHER EXPRESSED PROTEINS

Muscle biopsies from a myoblast transfer gene therapy clinical trial (Gussoni, E., et al., Na ture , 356:435-438 (1992) were re-analyzed to follow the fate of donor myoblasts at the single cell level with coincident detection of dystrophin or other proteins. Six DMD patients who participated in the trial had a molecularly characterized deletion in the dystrophin gene, which enabled the design of DΝA probes to the deleted area that specifically hybridized to donor cell nuclei but not the endogenous (patient) gene.

Immunohistochemistry was performed as described in Example 4, on serial sections to visualize either dystrophin or spectrin (a marker protein for muscle membrane) as sarcolemmal markers.

By using alternate consecutive sections immunostained for either dystrophin or spectrin, it was possible to determine whether any given donor nucleus was within a fiber and whether it produced dystrophin. Donor nuclei fused into host myofibers and expressing sarcolemmal dystrophin were detected in all samples. To estimate the proportion of donor nuclei remaining at one and six months after implantation, the volume analyzed for each sample was calculated by multiplying the area of serial section assessed microscopically by the number of sections examined and their thickness. The entire field in each serial section was scanned for the presence of donor nuclei. The number of donor nuclei detected in the analyzed volume was extrapolated to the entire injected volume and compared to the initial number of injected cells. For each patient, the estimated percentage of donor cells remaining at one month and six months after transplantation was consistently higher (3.1%-14.3%) than previously reported (about 1%, Gussoni, E., et al . , Nature, 356:435-438 (1992). In each serial section, the status of detected donor nuclei (mononuclear or fused in myofiber) was also noted. In the majority of patients, donor nuclei were seen fused to host myofibers. Only in two patients were more than half of the donor nuclei not fused into host myofibers. These results demonstrate that a proportion of donor nuclei gained access to host myofibers and persisted over time. Interestingly, in nearly all patients, a variable proportion of donor nuclei successfully fused to host myofibers failed to express dystrophin. In a separate experiment, to study whether a possible immune reaction to donor cells was occurring in the host, an anti-CD3 antibody which recognizes T-cells was applied (Boehringer Mannheim, Indianapolis, IN) . The antibody was applied to sections at a concentration of 10 micrograms/ml . FISH was subsequently performed, as described in Example 4, to allow the microscopic co- detection and visualization of the donor nuclei and infiltrating T-cells from the host. Among the six patients analyzed in this study, only one patient showed evidence of an immune response one month following myoblast injection. Donor nuclei detected by FISH were surrounded by host nuclei that were determined to be infiltrating T-cells by staining with anti-CD3 antibody.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method of simultaneously detecting, in a biological sample, the presence of a single copy of a exogenous target nucleic acid and the presence of a protein encoded by the target nucleic acid, comprising the steps of : a.) obtaining a preserved and sectioned sample containing cells into which the exogenous nucleic acid has been introduced; b.) contacting the sample with a primary antibody that is specific for the protein encoded by the exogenous nucleic acid, under conditions which allow specific binding of the primary antibody to the protein encoded by the exogenous nucleic acid; c.) contacting the sample with a nucleic acid probe, under conditions which allow specific hybridization of the nucleic acid probe to the exogenous nucleic acid; and d. ) simultaneously visualizing hybridization of the nucleic acid probe to the exogenous nucleic acid and binding of the primary antibody to the protein encoded by the exogenous nucleic acid, wherein hybridization of the nucleic acid probe to the exogenous nucleic acid is indicative of the presence of the exogenous nucleic acid in cells of the sample, and binding of the primary antibody to the protein encoded by the exogenous nucleic acid is indicative of the presence of the protein encoded by the exogenous nucleic acid in the sample.

2. The method of Claim 1, wherein the biological sample comprises mammalian cells or tissue into which the exogenous nucleic acid has been introduced.

The method of Claim 2, wherein the mammalian cells or tissue are human cells or tissue.

4. The method of Claim 1, wherein the nucleic acid probe is approximately 150-200 base pairs in final length.

The method of Claim 1, wherein the nucleic acid probe is labelled with a fluorochrome .

6. The method of Claim 5, wherein the fluorochrome is selected from the group consisting of: fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine isothiocyanate, Texas red, and phycoerythrin .

7. The method of Claim 1, wherein the nucleic acid probe is labelled for enzymatic detection.

8. The method of Claim 7, wherein the nucleic acid probe is labelled with alkaline phosphatase or horseradish peroxidase .

9. The method of Claim 1, wherein the nucleic acid probe is labelled with a nucleotide analog.

10 The method of Claim 9, wherein the nucleotide analog is digoxigenin.

11. The method of Claim 10, further comprising contacting the nucleic acid probe with an anti-digoxigenin antibody under conditions which allow binding of the anti-digoxigenin antibody to the digoxigenin in step b, and detecting hybridization of the nucleic acid probe to the exogenous nucleic acid by detecting the presence of the anti-digoxigenin antibody.

12. The method of Claim 11, wherein the anti-digoxigenin antibody is labelled with a fluorochrome .

13. The method of Claim 11, wherein the anti-digoxigenin antibody is labelled for enzymatic detection.

14. The method of Claim 1, wherein the primary antibody is labelled for enzymatic detection.

15. The method of Claim 1, wherein the primary antibody is labelled with a fluorochrome .

16. The method of Claim 15, wherein the fluorochrome is selected from the group consisting of: fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine isothiocyanate, Texas red, and phycoerythrin .

17. The method of Claim 1, further comprising contacting the primary antibody with a secondary antibody under conditions which allow binding of the secondary antibody to the primary antibody in step b, and -. detecting the presence of binding of the primary antibody to the protein encoded by the exogenous nucleic acid by detecting the secondary antibody.

18. The method of Claim 17, wherein the secondary antibody is labelled with a fluorochrome .

19. The method of Claim 18, wherein the fluorochrome is selected from the group consisting of: fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl 5 rhodamine isothiocyanate, Texas red, and phycoerythrin .

20. The method of Claim 17, wherein the secondary antibody is biotinylated. 0

21. The method of Claim 17, wherein the secondary antibody is labelled for enzymatic detection.

22. A method of evaluating the efficacy of a gene therapy 5 protocol in a host, comprising the simultaneous detection in a host cell or tissue of a single copy of an exogenous gene that has been introduced into a host cell or tissue and its protein product, whereby the detection of the presence of at least one copy of the 0 introduced gene, and its protein product, in the host cell or tissue is indicative of the efficacy of the gene therapy protocol in the host .

23. A method of evaluating the efficacy of a gene therapy protocol in a host comprising the steps of: a.) obtaining a preserved and sectioned sample containing host cells into which an exogenous gene has been introduced; b.) contacting the sample with a primary antibody that is specific for the protein encoded by the exogenous gene, under conditions which allow specific binding of the primary antibody to the protein encoded by the exogenous gene; c.) contacting the sample with a nucleic acid probe, under conditions which allow specific hybridization of the nucleic acid probe to the exogenous gene ; and d.) simultaneously detecting and visualizing hybridization of the nucleic acid probe to the exogenous gene and binding of the primary antibody to the protein encoded by the exogenous gene, wherein hybridization of the nucleic acid probe to a single copy of the exogenous gene is indicative of the presence of the exogenous gene, and binding of the primary antibody to the protein encoded by the exogenous gene is indicative of efficacy of the gene therapy protocol in the host.

24. A method of evaluating the efficacy of a gene therapy protocol in a host comprising the steps of : a.) obtaining a preserved and sectioned sample containing host cells into which an exogenous gene has been introduced; b.) contacting the sample with a primary antibody that is specific for the protein encoded by the exogenous gene, under conditions to allow specific binding of the primary antibody to the protein encoded by the exogenous gene, and contacting the primary antibody with a secondary antibody, under conditions which allow binding of the secondary antibody to the primary antibody; c.) contacting the sample with a nucleic acid probe labelled with a nucleotide analog, under conditions which allow specific hybridization of the nucleic acid probe to the exogenous gene, and contacting the nucleic acid probe with antibody to the nucleotide analog, under conditions which allow binding of the anti-nucleotide analog antibody to the nucleotide analog; and d.) simultaneously detecting hybridization of the nucleic acid probe to the exogenous gene by detecting the presence of anti-nucleotide analog antibody bound to the nucleotide analog, and detecting binding of the primary antibody to the protein encoded by the exogenous gene by detecting the presence of binding of the secondary antibody to the primary antibody, wherein hybridization of the nucleic acid probe to a single copy of the exogenous gene, and binding of the primary antibody to the protein encoded by the exogenous gene is indicative of the efficacy of the gene therapy protocol in the host .

25. The method of Claim 24, wherein the anti-nucleotide analog antibody is conjugated to a first fluorophore, and the secondary antibody that recognizes the primary antibody to the protein is conjugated to a second fluorophore .

26. The method of Claim 25, wherein the first fluorophore is rhodamine and the second fluorophore is fluorescein isothiocyanate, or the first fluorophore is fluorescein isothiocyanate and the second fluorophore is rhodamine.

27. The method of Claim 26, wherein hybridization of the nucleic acid probe to the exogenous nucleic acid , and binding of the primary antibody to the protein, are detected by immunofluorescence microscopy.

28. The method of Claim 27, wherein the nucleotide analog is digoxigenin.