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WO1998002577A1 - Proceder pour detecter conjointement des genes introduits et leurs produits - Google Patents

Proceder pour detecter conjointement des genes introduits et leurs produits Download PDF

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Publication number
WO1998002577A1
WO1998002577A1 PCT/US1997/010998 US9710998W WO9802577A1 WO 1998002577 A1 WO1998002577 A1 WO 1998002577A1 US 9710998 W US9710998 W US 9710998W WO 9802577 A1 WO9802577 A1 WO 9802577A1
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nucleic acid
antibody
exogenous
primary antibody
hybridization
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PCT/US1997/010998
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English (en)
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Louis M. Kunkel
Emanuela Gussoni
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The Children's Medical Center Corporation
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Priority to AU37177/97A priority Critical patent/AU3717797A/en
Publication of WO1998002577A1 publication Critical patent/WO1998002577A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • DMD Duchenne muscular dystrophy
  • PCR polymerase chain reaction
  • 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 .
  • 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.
  • 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.
  • introduction 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.
  • 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.
  • 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
  • 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).
  • 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.
  • 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.
  • 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,
  • 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 .
  • 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 .
  • 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 can be determined using the methods described herein.
  • exogenous gene localization and expression was investigated using two therapeutic protocols.
  • a plasmid herpes simplex virus vector (pHSVlac) expressing beta- galactosidase ⁇ j ⁇ -Gal) was used to deliver ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the exogenous DNA can be introduced in a vector, or via a genetically altered virus containing the exogenous nucleic acid of interest.
  • the exogenous DNA that is either absent from, or defectively produced in a target cell can be delivered via normal donor cells.
  • the exogenous DNA that is either absent from, or defectively produced in a target cell can be delivered via normal donor cells.
  • the exogenous DNA that is either absent from, or defectively produced in a target cell the "recipient cell”
  • DMD Duchenne 's Muscular Dystrophy
  • 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.
  • test sample 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 .
  • 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.
  • 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.
  • a non cross- linking fixative HistochoiceTM, Amresco, Solon, OH
  • 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 HistochoiceTM.
  • 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.
  • an appropriate fixative such as methanol
  • 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 ⁇ m (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 .
  • tissue 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 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” .
  • antibody 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 .
  • 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.
  • the primary antibody is detected with a secondary, anti-IgG antibody conjugated with FITC.
  • 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.
  • the prepared sample is fixed, using an appropriate fixative, such as HistochoiceTM, 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.
  • DAPI 4' -6'- diamidino-2-phenylindole
  • in-situ hybridization 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) .
  • 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) .
  • 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.
  • the probe 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) .
  • a reporter molecule e.g., biotin, digoxigenin
  • 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.
  • 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.
  • a reporter molecule such as digoxigenin
  • a reporter-binding molecule such as an anti-digoxigenin antibody
  • 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) .
  • 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.
  • FISH fluorescent in-situ hybridization
  • the target sequence is a nucleic acid of interest of approximately 5-40 kb in size.
  • 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.
  • the probe 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.
  • 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.
  • 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.
  • 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 .
  • 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 ⁇ g/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 .
  • 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) ) .
  • 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 ⁇ g 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 50 ⁇ l IX hybridization cocktail (50% deionized formamide, IX SSC (20X SSC: 3M NaCl, 0.3M Na 3 citrate2H 2 0, pH 7.0), 10% dextran sulfate, IX Denhardt ' s (100X Denhardt' s: 2% bovine serum albumin, 2% Ficoll, 2% polyvinylpyrrolidone) .
  • 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.
  • DAPI diamidino-2-phenylindole
  • EXAMPLE 2 FISH OPTIMIZATION
  • 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) .
  • 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.
  • the proportion of hybridization signals detected versus those expected was calculated as the percentage of the ratio between the number of positive hybridizing signals counted 131 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
  • 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.
  • 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.
  • 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 ⁇ -gal protein was detected, as shown by the absence of green staining.
  • 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 D ⁇ A, 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.
  • 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 D ⁇ A 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.

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Abstract

Cette invention se rapporte à des procédés pour détecter et simultanément visualiser, dans une cellule hôte, la présence d'une copie unique d'un acide nucléique exogène et d'une protéine codée par cet acide nucléique exogène.
PCT/US1997/010998 1996-07-15 1997-07-15 Proceder pour detecter conjointement des genes introduits et leurs produits WO1998002577A1 (fr)

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US8305579B2 (en) 2006-11-16 2012-11-06 Thomas Pirrie Treynor Sequential analysis of biological samples
US8822147B2 (en) 2006-11-16 2014-09-02 General Electric Company Sequential analysis of biological samples
US9201063B2 (en) 2006-11-16 2015-12-01 General Electric Company Sequential analysis of biological samples
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US7674589B2 (en) 1998-10-07 2010-03-09 Genentech, Inc. Methods for tissue analysis
US6573043B1 (en) 1998-10-07 2003-06-03 Genentech, Inc. Tissue analysis and kits therefor
US6905830B2 (en) 1998-10-07 2005-06-14 Genentech, Inc. Tissue analysis and kits therefor
US7129051B2 (en) 1998-10-07 2006-10-31 Genentech Inc Tissue analysis and kits therefor
US7344840B2 (en) 1998-10-07 2008-03-18 Genentech, Inc. Tissue analysis and kits therefor
US7468252B2 (en) 1998-10-07 2008-12-23 Genentech, Inc. Methods for tissue analysis
US7919254B2 (en) 1998-10-07 2011-04-05 Genentech, Inc. Tissue analysis and kits therefor
WO2000020641A1 (fr) * 1998-10-07 2000-04-13 Genentech, Inc. Analyse de tissu et necessaires a cet effet
US8091987B2 (en) 2005-07-07 2012-01-10 Xaar Plc Ink jet print head with improved reliability
US11413296B2 (en) 2005-11-12 2022-08-16 The Regents Of The University Of California Viscous budesonide for the treatment of inflammatory diseases of the gastrointestinal tract
EP1991708A4 (fr) * 2006-03-13 2009-12-02 Ikonisys Inc Procede permettant de combiner immunocoloration et hybridation en fluorescence in situ (fish) au moyen de fluorophores pouvant se lier de manière covalente
US8305579B2 (en) 2006-11-16 2012-11-06 Thomas Pirrie Treynor Sequential analysis of biological samples
US8822147B2 (en) 2006-11-16 2014-09-02 General Electric Company Sequential analysis of biological samples
US9201063B2 (en) 2006-11-16 2015-12-01 General Electric Company Sequential analysis of biological samples
US9518982B2 (en) 2006-11-16 2016-12-13 General Electric Company Sequential analysis of biological samples
WO2010022332A1 (fr) 2008-08-22 2010-02-25 Ventana Medical Systems, Inc. Procédé pour la détection chromogénique d’au moins deux molécules cibles dans un unique échantillon
US8481270B2 (en) 2008-08-22 2013-07-09 Ventana Medical Systems, Inc. Method for chromogenic detection of two or more target molecules in a single sample
US9677125B2 (en) 2009-10-21 2017-06-13 General Electric Company Detection of plurality of targets in biological samples

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