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WO1997013869A1 - Detection de mutations specifiques d'alleles par amplification pcr in situ a transcriptase inverse, specifique d'alleles - Google Patents

Detection de mutations specifiques d'alleles par amplification pcr in situ a transcriptase inverse, specifique d'alleles Download PDF

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Publication number
WO1997013869A1
WO1997013869A1 PCT/US1996/016162 US9616162W WO9713869A1 WO 1997013869 A1 WO1997013869 A1 WO 1997013869A1 US 9616162 W US9616162 W US 9616162W WO 9713869 A1 WO9713869 A1 WO 9713869A1
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dna
cells
allele
seq
type
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PCT/US1996/016162
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English (en)
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Deiter C. Gruenert
Austin Dohrman
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The Regents Of The University Of California
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Priority to AU72626/96A priority Critical patent/AU7262696A/en
Publication of WO1997013869A1 publication Critical patent/WO1997013869A1/fr

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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention concerns a method and an assay for detection and differentiation between expression of unmutated wild-type DNA sequences and between endogenous DNA sequences in vi tro or in vivo .
  • the invention concerns the method and the allele-specific in si tu reverse transcriptase polymerase chain reaction (RT- PCR) amplification assay for detection and differentiation between expression of unmutated wild-type DNA sequences and between endogenous mutant DNA sequences or visa versa.
  • the method is useful for verification, diagnostic assessment and monitoring of therapeutic small fragment homologous replacement gene therapy, for diagnostic assessment and - monitoring of cDNA-based gene therapies, for analysis of gene expression of specific alleles during fetal development and for diagnostic assessment of the expression of alleles involved in cancer mutations.
  • ADA adenosine deaminase deficiency
  • CF cystic fibrosis
  • the SFHR method when applied in vivo is useful for gene therapy treating human genetic diseases and for countering the deleterious effects of these diseases.
  • this method circumvents inappropriate cell expression and the physiological and metabolic complications that result.
  • One aspect of the current invention is a method for detection of expression and for differentiation between
  • Another aspect of the current invention is a method for detection and differentiation of expression of endogenous mutated DNA and exogenous wild-type DNA using an assay comprising a reverse transcriptase polymerase chain reaction amplification of mRNA-derived DNA, cDNA or DNA fragment sequences.
  • Yet another aspect of the current invention is a method for detection and differentiation of expression of endogenous mutated DNA and exogenous wild-type DNA with an allele-specific detection of expressed gene using allele-specific primers.
  • Still another aspect of the current invention is a method for detection and differentiation of expression of endogenous mutated DNA and exogenous wild-type DNA using the first allele-specific primer for endogenous mutated DNA sequence, the second allele-specific primer for therapeutic nonmutated wild-type DNA sequence and the non-allele specific third primer specific for a DNA sequence in an exon that is outside of the region of homology defined by the wild-type therapeutic DNA.
  • Another aspect of the current invention is an allele-specific primer specific for an endogenous mutated or nonmutated gene.
  • Another aspect of the current invention is an allele-specific primer specific for a therapeutic nonmutated wild ⁇ type DNA fragment.
  • Another aspect of the current invention is an mRNA-specific primer for the reverse transcriptase reaction from DNA to cDNA.
  • Still another aspect of the current invention is a method useful for evaluation of gene therapy protocol, and for verification, management and determination of efficacy of gene therapy.
  • Still yet another aspect of the current invention is - a method for diagnosis of genetic diseases during fetal development by detecting expression of the mutant allele in fetal cells.
  • Still another aspect of the current invention is a method for the diagnostic assessment of the expression of mutant alleles involved in cancer comprising detection of cancerous mutations present in precancerous cells having a normal phenotype.
  • Another aspect of the current invention is a diagnostic assay for detection of and differentiation between gene expression in tested tissue which is mutated or nonmutated, and gene expression in the same type normal tissue, which method comprises steps:
  • step (c) digesting the cells of the sample of step (b) to expose the single strand mRNA and eliminate DNA contained in the cells;
  • step (d) subjecting the mRNA of step (c) to reverse transcription reaction to obtain first-strand complementary DNA (cDNA) from the mRNA template of the sample of cells of step (a) ;
  • step (e) subjecting the cDNA of step (d) to polymerase chain reaction amplification using allele-specific primers for tested tissue and a solution comprising all necessary nucleotides in sufficient quantity to obtain the cDNA in sufficient amount for the assay, wherein at least one nucleotide in the solution or in the primer is labelled with a non-interfering radioactive, immunochemical or fluorescent marker detectable by spectroscopic, autoradiographic emulsion, immunocytochemical or enzymatic detection means;
  • step (f) obtaining a sample of the tissue cells of the same type of nonmutated tissue as in step (a) or tissue submitted to gene therapy or a sample of wild-type DNA fragment corresponding to nonmutated normal DNA;
  • step (g) fixing the cells of step (f) ;
  • step (h) digesting the cells of the step (g) to expose the single strand mRNA and eliminate DNA contained in the cells;
  • step (i) subjecting the mRNA of step (h) to reverse transcriptase reaction to produce first-strand complementary DNA (cDNA) from the mRNA template of the cells of step (h) ;
  • step (j) subjecting the cDNA produced in step (i) to polymerase chain reaction conditions using allele-specific primers for nonmutated tissue a solution comprising the necessary nucleotides, with the proviso that at least one nucleotide is labelled with a non-interfering radioactive, immunochemical or fluorescent marker which is detectable by spectroscopic means, immunoreaction means, autoradiographic means or by enzymatic detection means; and
  • step (k) comparing the results obtained in step (e) with the results obtained in step (j) and observing the presence or absence of the detectable marker produced in step (j) in cDNA of step (e) to determine quantitatively or qualitively the presence of allele-specific mutation in the endogenous gene expression using spectroscopic, autoradiographic or enzymatic detection means.
  • Another aspect of the current invention concerns an assay method to verification, management and assessment of efficacy of gene therapy using differentiation between gene expression in tested mutated tissue which has been subjected to gene therapy and gene expression in the similar or the same type of normal tissue, which method comprises : (a) obtaining a sample of the tested tissue cells subjected to gene therapy using small fragment homologous replacement of the endogenous DNA with the wild-type DNA with a sample of wild-type DNA fragment corresponding to the nonmutated normal DNA; (b) fixing the cells of step (a) ;
  • step (c) digesting the cells of step (b) to expose the single strand mRNA and eliminate DNA contained in the
  • step (d) subjecting the mRNA of step (c) to reverse transcription reaction to obtain first-strand complementary
  • DNA from the mRNA template of the sample of cells of step (a) ;
  • step (e) subjecting the cDNA of step (d) to polymerase chain reaction amplification in the presence of allele- specific primers for tested tissue using a solution comprising all necessary nucleotides in sufficient quantity to produce the cDNA in sufficient amount for the assay, wherein at least one nucleotide in the solution or in the primer is labelled with a non-interfering radioactive, immunochemical or fluorescent marker detectable by spectroscopic means, immunocytochemical means, autoradiographic emulsion or by enzymatic detection means;
  • step (f) obtaining a sample of normal nonmutated tissue cells ; (g) fixing the cells of step (f) ;
  • step (h) digesting the cells of the step (g) to expose the single strand mRNA and eliminate DNA contained in the cells;
  • step (i) subjecting the mRNA of step (h) to reverse transcriptase reaction to produce first-strand complementary DNA (cDNA) from the mRNA template of the cells of step (h) ;
  • step (j) subjecting the cDNA produced in step (i) to polymerase chain reaction conditions using allele-specific primers for nonmutated tissue and a solution comprising the necessary nucleotides, with the proviso that at least one nucleotide is labelled with a non-interfering radioactive, enzymatic, immunocytochemical or fluorescent marker which is detectable by spectroscopic means, immunochemical means, autoradiographic means or by enzymatic detection means; and
  • step (k) comparing the results obtained in step (e) with the results obtained in step (j) and detecting in the sample of step (e) the presence or absence and a quantity of at least one labeled marker present in the DNA produced
  • Still yet another aspect of the current invention is an assay useful for detection of the expression of mutant genes during fetal development or for verification of successful gene therapy using small fragment homologous replacement in correction of the mutated gene associated with cystic fibrosis, Fanconi's anemia, sickle cell anemia, retinitis pigmentosa, xeroderma pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, Duchenne's muscular dystrophy, or Tay-Sachs disease.
  • BRIEF DESCRIPTION OF DRAWINGS Figure 1 is a schematic representation of the PCR analysis of genomic CFTR DNA amplifying the genomic locus containing the region targeted for SFHR of the ⁇ F508 mutation.
  • Figure 2 represents a strategy for RT-PCR analysis of CFTR mRNA.
  • Figure 3 is allele-specific PCR analysis of transfected ⁇ CFTE29o- cells using primers CF7B/CF6 (N) or
  • Figure 3A shows primer pair CF8B/CF6 resulting in 411 bp fragment.
  • Figure 3B shows primer pair CF7B/CF6 resulting in 414 bp fragment.
  • Figure 4 is allele specific PCR analysis of CFPAC-1 cells with primers CF1A/CF7C transfected with rec A coated and uncoated 491 base fragments.
  • Figure 5 shows RT-PCR analysis of CFTR mRNA from HTE- 11 cells transfected with uncoated or coated 488-nt fragments in a dendrimer-DNA complex.
  • Figure 6 is an autoradiographic analysis of DNA amplified from first-strand CFTR cDNA from ⁇ CFTE29o- cells.
  • Figure 7 illustrates restriction enzyme digestion analysis of the allele-specific amplification product generated with primers CF1B/CF8C from PCR amplification of DNA from nontransformed primary airway epithelial cells -transfected with 488 base fragments containing the ⁇ F508 mutation and a unique Xhol restriction site.
  • Figure 8 is a schematic representation of the RT-PCR and Dde I restriction digestion analysis of 3-globin mRNA- derived cDNA.
  • Figure 9 are fluorescent micrographs of allele- specific in situ RT-PCR of cell lines.
  • Figure 12A shows the result of in si tu PCR of 16HBE14o " cells following DNAse treatment and reverse transcription (+rt) . Only this micrograph is positive for the presence of wtCFTR in mRNA. The other 3 micrographs are negative.
  • Figure 12B shows the result of in si tu PCR of ⁇ CFTE29o- cells after reverse transcription.
  • Figure 9C shows the result of in si tu PCR of 16HBE14o- without reverse transcription.
  • Figure 9D shows the result of in si tu PCR of ⁇ CFTE29o- without reverse transcription.
  • Figure 10 is an fluorescent micrograph of cells which show allele-specific in si tu RT-PCR of a section of normal bronchial epithelial tissue.
  • Figure 11 is an allele-specific in si tu RT-PCR in human tissue.
  • Figure IIA shows wild-type CFTR mRNA detected in normal bronchial tissue.
  • Figure 11B shows wild-type CFTR mRNA detected in normal human colon tissue.
  • Figure 12 is allele-specific in si tu RT-PCR in human bronchial epithelial cell line.
  • Figure 15A shows wild-type CFTR mRNA detected in normal bronchial epithelial cells using a wild-type specific oligonucleo-tide primer pair.
  • Figure 15B shows detection of mutated CFTR mRNA in cystic fibrosis bronchial epithelial cells.
  • Figure 12C shows a detection of mRNA in mixed population of mutated and nonmutated wild-type cells.
  • Figure 13 shows allele-specific RT-PCR analysis of RNA samples isolated from transfected mice at day 4 post- transection in various organs transfected or nontransfected ( Figure 13A) and Kpn 1 Restriction enzyme cleavage ( Figure 13B) .
  • Allele means one of two or more alternate forms of a gene occupying the same locus on a particular chromosome. Alleles that functions normally are the wild ⁇ type alleles. Mutated alleles means alleles that function abnormally in comparison with the wild-type alleles. "Wild-type or wt genomic DNA” means normal genomic DNA that does not contain a site that alters the amino acid sequence of the normal protein product and is not associated with a disease or dysfunction.
  • “Mutated DNA” means altered DNA sequence within a gene that results in a phenotype abnormality and causes and is manifested as a disease or dysfunction.
  • Exogenous DNA means the altering DNA used for changing endogenous genomic DNA sequences by small fragment homologous replacement.
  • This DNA can be wild-type DNA used to correct a mutant gene or the mutant genomic DNA used to induce the mutation, for example, in transgenic animals serving as an animal model for study of the genetic disease, or the wild-type DNA serving as a standard for qualitative and quantative assessment of gene mutations or correction thereof by gene therapy.
  • Exogenous DNA is used as a control
  • Endogenous DNA means, for diagnostic purposes, a tested genomic DNA sequence present in the native gene, or for therapeutic purposes, the mutated DNA sequence to be altered by gene therapy.
  • This DNA can either be cellular genomic DNA or pathogen genomic DNA. This DNA sequence is resident within a target cell for diagnosis or therapy.
  • Gene therapy means treatment of a patient suffering from a disease resulting from expression of genetic material that is mutated, i.e. its nucleotide sequence is different from the nucleotide sequence of a normal individual . Gene therapy thus means replacement of the dysfunctional gene with a functional gene.
  • RT or reverse transcription means mRNA directed DNA synthesis.
  • the technique is described in J. Biol. Chem. , 260:9326 (1985) , hereby incorporated by reference.
  • PCR or "polymerase chain reaction” means an enzymatic in vi tro amplification of DNA sequences. The technique, described in Science, 239:1350 (1988) is hereby incorporated by reference.
  • RT-PCR means reverse transcriptase polymerase chain reaction.
  • the reverse transcription polymerase chain reaction is according to the description of the art found in, for example, in U.S. Patents 5,416,192; 5,416,260 5,418,134; 5,418,149; 5,418,162; 5,420,009; 5,422,242 5,424,184; 5,424,189; 5,426,026; 5,426,039; 5,427,909 5,427,929; 5,427,932; 5,434,048; 5,435,309; 5,436,142 5,436,144; 5,436,149; and 5,436,326, all of which are incorporated herein by reference.
  • the current invention concerns a method and an assay which detects and differentiates between expression of therapeutic, normal nonmutated wild-type DNA and between mutated DNA in vi tro or in vivo .
  • the invention is useful for verification and assessment of gene therapy and in diagnostics.
  • the assay involves allele-specific in si tu reverse transcriptase polymerase chain reaction (RT-PCR) amplification of endogenous, typically mutated DNA and of exogenous, normal wild-type DNA or RNA. Expression of each of these DNAs and/or RNA is detected using labeled nucleotide marker or its precursor in the samples of the same cell types and results are qualitatively and quantatively compared to determine whether the mutated or normal DNA is completely, substantially or predominately expressed.
  • RT-PCR si tu reverse transcriptase polymerase chain reaction
  • SFHR therapy comprises identifying certain typically mutated sequence within genomic DNA, obtaining and introducing (in vivo or . in vitro) into cells that contain the mutated sequence a population of small endogenous or wild-type therapeutic DNA fragments that contain both exon and flanking noncoding sequences. These small fragments of exogenous DNA which are able to correct mutant genomic DNA sequences by homologous replacement, are expressed in the cells. The expressed exogenous DNA as mRNA and ultimately as protein leads presumably to correction of the dysfunction caused by the mutated gene. Until today, however, no method was available to verify and/or quantify the successful replacement of mutated DNA with therapeutic nonmutated DNA.
  • the invention is also useful for designing and testing the gene therapy protocol before the gene therapy is used, and for testing and designing allele-specific and allele-non-specific primers.
  • the invention is useful for a variety of diagnostic assays for monitoring expression of disease genes in specific tissue.
  • the invention is useful for detection of the expression of mutant alleles during fetal development or for detection of precancerous cells having a normal phenotype but carrying cancer mutations.
  • tissue is obtained from the tested individual typically suffering from the genetic disease or having family history or other predisposition to genetic disease, cancer, etc.
  • tissue is obtained from the tested individual typically suffering from the genetic disease or having family history or other predisposition to genetic disease, cancer, etc.
  • cystic fibrosis human bronchial or lung biopsies, or cells or cell lines from normal patients (wild-type DNA) and from cystic fibrosis patients (mutated DNA) are obtained or in case of, for example, skin melanoma, the healthy skin cells
  • Site of the mutation is derived from the difference between the normal (wild type) sequence and the sequence obtained from the individual suffering from the disease controlled by that particular gene.
  • the most common mutation ⁇ F508 of the cystic fibrosis transmembrane conductance regulator (CFTR) is found in exon 10, and results in a phenylalanine deletion in the CFTR protein.
  • the mutation causing sickle cell anemia is caused by an A to T transversion in the sixth codon of the human ⁇ -globin gene resulting in a glutamine to valine substitution in the protein.
  • the mutation causing xeroderma pigmentosum group G is due to a deletion of an A in a run of AAA of the exon containing the mutation, as well as mutated sequences.
  • wild-type DNA sequence homologous to the site of the mutation but not containing the mutation is obtained.
  • the wild-type sequence may be isolated or synthesized.
  • the synthesis of the short homologous DNA fragment (wild-type DNA fragment) may be conducted by methods known in the art, such as the isolation and separation of a wild-type DNA fragment by cleavage with restriction endonuclease, PCR amplification, de novo oligonucleotide synthesis, and/or combinations of enzyme restriction and ligation to produce deletions, additions or the like.
  • Wild-type DNA fragments of the region where the DNA mutation occurs are isolated, for example, as described in Example 1 and the allele-specific primers are prepared to that particular region.
  • Allele-specific primers are prepared to the endogenous mutated DNA or RNA. Additionally, primers are prepared which are non-allele specific. These primers are specific for a DNA sequence in an exon other than the exon containing the mutation and are outside of the region of homology which is defined by the therapeutic or by the normal wild-type DNA fragment. Additionally, primers for
  • primers contain sequences complementary to the mRNA as well as sequences that are unique and not specific for any mRNA sequences.
  • PCR primer is based on the unique sequence found in the reverse transcription primer.
  • the cells are grown to about 70% confluence and are trypsinized, washed, for example, drops or in phosphate buffer saline and resuspended in the same buffer, as cytospins on slides, such as Superfrost plus slides, obtained from Fisher Company or Perkin Elmer.
  • Cells are fixed in any suitable fixation solution, such as about 4% paraformaldehyde in IX PBS (PFA) and stored at about -80°C until used. In the alternative, they are used without freezing.
  • Cells fixed on slides are submitted to heating at about 35-50°C, preferably 55°C for about 10-30 minutes to bind the cells to the slides.
  • Cells are then digested, and/or permeabilized for example, with trypsin pepsin 2 mg/ml in 0.1 N HCl (0.01-0.05%) , pronase (l-10 ⁇ g/ml) or other digestive enzyme, or chemical agents, such as 1.5MNall, O.lMNaOH 1 , 70% EtOH or 0.5% NP-40 for a time which is dependent on length of time of fixation, typically for about 5-45, preferably 10-30 minutes.
  • tissue or cells are permeabilized, mRNA is exposed and proteins, enzymes, etc., which could interfere with PCR are destroyed.
  • the sample is treated in RT buffer, such as, for example, Perkin Elmer reverse transcriptase buffer, dithioerythriol (DTT) , each nucleotide (0.5-lmM) , primers
  • RT buffer such as, for example, Perkin Elmer reverse transcriptase buffer, dithioerythriol (DTT) , each nucleotide (0.5-lmM) , primers
  • RNasin (1 - 2.5 ⁇ l) and reverse transcriptase and 2500 ⁇ /ml (about l ⁇ l) .
  • the solution is covered and placed in a humid chamber at 30-50°C, preferably at 42°C, for about 10-120 minutes, preferably for 45 minutes, and reverse transcribed.
  • the samples are 15 then washed with PBS.
  • all the above steps such as fixation, digestion, permeabilization, reverse transcription and polymerase chain reaction can be performed using other agents and conditions as known now or will become known in the future as long as they in general can perform functions, as described.
  • the proper controls must be included in the assay. All three sections or cell drop spots as described above, must be on the same slide.
  • the tested section is treated with both DNase and is reverse transcribed (RT) .
  • the other two sections on the slide are the positive control (-DNase, +RT) and the negative control (+DNase, -RT) .
  • the positive control should show staining strongly in the nucleus, the negative control should show no staining.
  • the tested sample should show, if positive, the marker present in the cytoplasm.
  • DNase digestion is necessary to remove genomic DNA which would interfere with detection of mRNA by RT-PCR because the genomic DNA can incorporate labeled nucleotide and would result in false positive or false negative results.
  • DNase digestion achieved for example, with lU/ul in IM sodium acetate pH 5, 5 mM MgS0 4 is typically done overnight at 30-50°C, preferably at 37°C on two of the three sections, followed by a wash and dehydration step.
  • the reverse transcription step is performed on one of the DNased sections and on the non-DNased section, typically in 50 ul per sample using Perkin Elmer solutions, containing IX RT buffer, ImM DTT, ImM each dNTP, luM antisense primer or poly dt, 2500 u/mL M-MLV, or Superscript II, BRL 2000 u/mL. Parafilm coverslips are added, samples are placed in a humid chamber at 42°C for 45 minutes and washed.
  • the PCR reaction is done on all sections in 50 ul per section in a 50 ul final volume; lx PCR buffer II, 200 uM each nucleotide, 10 uM each primer, 4.5mM MgCl 2 , lOuM fluorescein labelled d-UTP (Boehringer mannheim) , and 10 units of Taq (Is) DNA polymerase are added.
  • a hotstart is performed with the Perkin Elmer in si tu PCR system 1000 thermal cycler; 25 cycles, target temperature, 59°C. Slides are subsequently washed in 0.1 X SSC, at 42-55°C for about 20 minutes to remove excess nucleotides.
  • This technique may be modified for various tissue materials by replacing fluorescein d-UTP with any of the following modified dNTPs: 10 uM digoxigenin or biotin-11- dUTP, or radioactively labelled d-UTP -either 33 P or 35 S.
  • the bioptic tissue of the treated individual is treated according to the invention, using wild-type allele-specific primers for detection of expression of normal nonmutated wild-type DNA. If gene therapy was successful, then there is fluorescence or radioactivity detected with these wild-type allele-specific primers. Quantitation of the expression in the cells is by counting the cells expressing the normal or mutated DNA or by using histograms or signal images, as described below.
  • Slides are viewed for fluorescence using any suitable fluorescent microscope, such as the Zeiss axiophot fluorescent microscope (450-490 nm wave length fluorescent epilumination) or a confocal microscope. Relative fluorescence per pixel of image may be compared using the confocal's histogram function. To determine fluorescent staining intensity of digitized grey scale images of cells or tissues captured in photoshop from the Zeiss microscope the NIH image program may be used.
  • immunocytochemistry is carried out using standard protocols and anti-dig or anti-biotin antibody (Fab fragment, alkaline phosphate conjugate. Briefly, slides are incubated in blocking buffer for 30 minutes at room temperature and washed in 0.1 M Tris-HCIpH7.9, 150 mM NaCl. About 50 ul of anti-dog or anti-biotin antibody diluted
  • autoradiography is carried out by dipping the slides in a photographic emulsion, such as Ilford K5D. Slides, stored in the dark at 4°C are developed at various times (1 to 5 days) in Kodak developers and fixers. Autoradiographic grains are viewed in either dark or bright field using any suitable microscope such as the Zeiss axiophot microscope that detects fluorescence. Obtained labeled DNA sequences are then compared for expression of mutated and/or wild-type DNA by following the presence or absence of the fluorescent, radioactive or chemical marker, as described above, for a qualitative assessment of gene therapy efficacy and quantitated by determining the number of cells expressing therapeutic RNA.
  • a photographic emulsion such as Ilford K5D. Slides, stored in the dark at 4°C are developed at various times (1 to 5 days) in Kodak developers and fixers. Autoradiographic grains are viewed in either dark or bright field using any suitable microscope such as the Zeiss axiophot microscope that detects fluorescence. Obtained labele
  • Confirmation of the allele specific RT-PCR technique is done by using conventional in si tu hybridization techniques using preferably non-radioactive or radioactive DNA or RNA probes. After the PCR step, slides are washed and a labelled probe is applied in a hybridization solution for a number of hours.
  • the hybridization buffer contains generally, 50% deionized formamide, 4xSSC, 10% dextran sulfate, lx Denhardt's solution, 250 ug/ml tRNA. The slides are washed in stringent buffers, (O.lx SSC at 45-
  • the assay and method of the invention were developed and tested on cystic fibrosis mutated and wild-type sequences.
  • the method is, however, equally applicable to other sequences.
  • Accurate detection of cystic fibrosis transmembrane conductance regulator (CFTR) expression on a cell-by-cell basis has greatly facilitated understanding of its distribution in tissue. Not only does cell specific detection of CFTR help define the distribution, it is useful for assessment of gene therapy efficacy and for verification of gene therapy occurrence.
  • One problem in gene therapy, as described above, has been limitations in the direct detection of cells which are expressing the correcting genetic material. At the present time, there are no antibodies that differentiate wild-type (wt) CFTR from mutant CFTR. Even the most common CFTR mutation, the ⁇ F508, cannot be distinguished from wtCFTR protein immunocytochemically. The same is true for verification of other gene replacement.
  • the approach utilized in this invention is to evaluate expression of CFTR or expression of any other mutation at the level of mRNA.
  • the invention utilizes allele-specific reverse transcriptase polymerase chain reaction (RT-PCR) amplification in solution. This approach has not previously been directly applied to in si tu analysis of cells in culture or in tissue sections.
  • fluorescent nucleotide and allele-specific oligonucleotide primers are utilized to differentiate wtCFTR from mutated ⁇ 508CFTR mRNA expression.
  • ⁇ F508 homozygote airway epithelial cell lines ( ⁇ CFTE29o- and CFBE4lo-) , it is possible to distinguish between cells expressing wtCFTR and ⁇ F508CFTR. In addition, it is possible to distinguish the tissue sites of CFTR mRNA expression.
  • the importance of the method and assay of the invention for gene therapy is that it enables identification, both qualitative and quantitative, of the cells expressing the wild-type therapeutic DNA and distinguishes them from mutated cells expressing mutated DNA.
  • the initial denaturation of the cells and cell tissue is conducted at 94-95°C for about 3 min.
  • the second denaturation is performed at 94-95°C for about 1 min.
  • the annealing is performed at target temperature 59°C for about 2 min.
  • the initial elongation is performed at 72°C for 1 min.
  • the final cycle elongation is performed at 72°C for 20 min.
  • Usually 25 cycles of PCR are needed to obtain a useful amount of cDNA.
  • the conditions, as described above, are modified for other cells or cells tissue, as needed. These modifications are within the skills of artisan.
  • the present invention provides a method for verification, assessment of efficacy and monitoring of gene therapy performed to correct defects associated with genetic human diseases, such as cystic fibrosis, thalassaemias, sickle cell anemia, Fanconi's anemia, retinitis pigmentosa, Xeroderma pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma,
  • genetic human diseases such as cystic fibrosis, thalassaemias, sickle cell anemia, Fanconi's anemia, retinitis pigmentosa, Xeroderma pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma,
  • CFTR CF transmembrane conductance regulator
  • Primers including allele-specific primers to the CF mutation are listed in Table 1.
  • a sickle cell anemia ⁇ -globin gene is mutated at the sixth amino acid from the amino terminus of the ⁇ chain by replacement of glutamic acid with valine.
  • the sickle cell hemoglobin contains the alteration in the / S-globin primary structure which places an aberrant hydrophobic amino acid on the surface of the protein, causing deoxygenated hemoglobin to aggregate, resulting in alteration in the red blood cells shape and impedance of blood flow through capillaries and small venules.
  • Primers including allele- specific primers are listed in Table 2.
  • Thalassaemias the human genetic diseases, are caused by a failure to synthesize adequate amounts of hemoglobin o. or ⁇ polypeptides.
  • mutations that create stop codons in the middle of the coding sequence prevent completion of the translation of mRNA, in others, mutations prevent normal intron splicing and therefore result in untranslatable mRNAs.
  • complementation group G (XP- G) , a 245 bp exon, contains a deletion of an adenosine (A) in a run of three adenosine (AAA) of bp 19-21 of the exon.
  • A adenosine
  • AAA adenosine
  • UV ultraviolet
  • Exposure to UV light results in skin cancer due to defective DNA repair.
  • XP-G this defect is associated with an abnormal endonuclease.
  • XP is often associated with central nervous system (CNS) defects.
  • Primers including allele-specific primers for the XP-G complementation group are listed in Table 3.
  • the assay of the invention is also useful for detection of mutations in fetal and other tissue as well as for early - diagnosis of potential dysfunctions and disturbances caused by DNA mutations.
  • the assay therefore, is used in detecting mutated genes in fetal tissue, for ammioanalysis and other tests typically used for early detection of the genetic disease.
  • the sample of the fetal tissue or amniotic fluid is obtained, mRNA of the suspected gene is obtained as described above and the presence or absence of the mutated DNA is determined by comparison with the wt DNA. If the normal wt DNA is expressed than the genetic disease is not present. If the normal wt DNA is not expressed or when the mutated DNA is expressed then the genetic disease is or will be present or there is a danger that it might develop.
  • the suspect tissue of precancerous cell or cells from the predisposed patients is submitted to the assay of the invention and the cancer mutation is either confirmed or not found. Early detection of precancerous cells provides an opportunity for early detection.
  • Allele-Specific Primers Used in the Assay of the Invention For Detection of Allele-Mutations The primer pairs used for PCR analysis were designed to be specific for the genomic locus in the regions where the mutation occurs.
  • the primers are allele-specific to the genomic locus in the region of exon 10 that contains the homologous region. Primers indicated in Figures 1 and 2 are defined in Tables 1 and 2.
  • oligo N (A) 5' -CACCAAAGATGATATTTTC-3 ' SEQ ID NO: 13 oligo ⁇ F (A) 5' -AACACCAATGATATTTTCTT-3 ' SEQ ID NO: 14
  • Classical sickle cell (SC) anemia is a disease caused by an A to T transversion in the sixth codon of the human beta-globin gene resulting in a Glu to Val substitution in the protein. Phenotypically there is a polymerization of the hemoglobin that results in a myriad of pathologies which ultimately lead to the death of the individual.
  • the disease is subject to gene therapy and the success of the gene therapy is determined by the assay of the invention.
  • Primers SC5 and SC6 are used to assay mRNA expression of the DNA that has undergone homologous replacement .
  • Primers SC-BA and SC-BS are allele-specific and differentiate between wild-type (SC-BA) and sickle (SC-BS) -globin sequences.
  • the (+) and (-) designate sense and antisense sequences, respectively.
  • Xeroderma pigmentosum Another disease which can be treated by homologous recombination using small fragments in xeroderma pigmentosum, a disease of skin.
  • Xeroderma pigmentosum is a rare, disfiguring syndrome inherited as an autosomal recessive trait.
  • Table 4 lists DNA sequences primers used for generation of short homologous fragments in the region of a 245 bp exon of complementation group G.
  • Allele-specific analysis of the wild-type (N) sequences of the XP group G gene is detected by amplification with either primers XP3/XP6A (237-bp) and XP7A/XP4 (374-bp) .
  • the XP G mutation (AAA>AA) was assayed by amplification with primers XP3/XP6B (237-bp) and XP7B/XP4 (374-bp) .
  • Sense (+)
  • antisense (-) .
  • Allele-specific primers XP6A and XP7A detect normal (N) sequences and XP6B and XP7B detect the XP-G mutation.
  • XP6A and XP6B are used in conjunction with XP3 to generate a fragment of 237-bp, while XP7A and XP7B are used with XP4 to give a product of 374-bp. Because the 374-bp fragment contains a Kpn I cut site, cleavage results in 97-bp and 277-bp restriction fragments.
  • Non-allele-specific primers for XP-G exon sequences will be determined from published information. For studies performed in vivo in mice, specific mouse murine SFTR cDNA primers were prepared. These primers are shown in Table 5.
  • Murine CFTR cDNA Primers mCFllR CTTGTGGGAAATCCTGTGCTGAA SEQ. ID NO: 47 (exon 11) w/mCF508-3 mCF12R CCTTCTCCAAGAACTGTGTTGTC SEQ. ID NO: 48 exon 11 mCF20R GGCTCTTAGGAAGAACTGGATCAGG SEQ. ID NO: 49 exon 20 mCF24R TTTCAGAGCAGTAATTTGCGTCCG SEQ. ID NO: 50 exon 24 mCF508(-) ATCGGTGTTTCCTATGATGAGTAC SEQ. ID NO: 51 exon 10 mCF508-2 (-) ATAGGAAACACCGATGATAT SEQ.
  • RNA primers and specific conditions for PCR are seen in Table 6.
  • Figure 1 is a schematic representation of the PCR analysis of genomic DNA.
  • Primers CF1B and CF6 are outside the region of homology and amplify only DNA derived from genomic DNA around the region of homology.
  • Primers CF7B and CF8B are sense (+) allele specific primers for wt and ⁇ F508 CFTR sequences, respectively. In combination with CF6 (-) these primers give rise to a fragment that is either 414 bp or 411 bp that represent the presence of wt or ⁇ F508 CFTR sequences, respectively. Their usefulness as allele-specific primers is exemplified in Figures 3 and 4.
  • the CF7C and CF8C primers are antisense (-) and are allele-specific for wt and ⁇ F508 CFTR sequences, respectively.
  • they In conjunction with the sense (+) primer CF1B, they amplify fragments that are 391 bp or 389 bp wt- or ⁇ F508-specific, respectively.
  • the restriction analysis with Xho I of the CF1B/CF7C or CF8C amplification product gives rise to two different restriction fragments of 283 bp and 109 bp (N) or 106 bp ( ⁇ F) .
  • Their usefulness as allele- specific primers is exemplified in Figure 7. If a secondary amplification is carried out with primers CF1/CF7C or CF8C, then the restriction fragments will be 199 bp and 109 bp (N) or 106 bp ( ⁇ F) .
  • Figure 2 is a schematic representation of the PCR analysis of genomic DNA illustrating strategy for RT-PCR analysis of CFTR mRNA by spanning intron/exon boundaries.
  • the resultant mixture of 473 bp (N) or 470 bp ( ⁇ F) fragments was then purified and reamplified by allele- specific PCR with primers CF17/CF7C OR CF17/CF8C.
  • Their usefulness as allele-specific RT-PCR primer pair are exemplified in Figures 3 and 4.
  • the resultant 330 bp (n) or 327 bp ( ⁇ F) fragments were digested with Xho I to
  • the restriction fragments were either 221 bp and 109 bp (nonCF) and 221 bp and 106 bp ( ⁇ F508) .
  • Figure 3 shows allele-specific PCR analysis (using primers CF7B/CF7 (N) or CF8B/CF7 ( ⁇ F) ) .
  • Figure 3 is an analysis of ⁇ CFTE290- cells transfected either with the StarburstTM dendrimer-DNA complex (lanes b and c) or the gramicidin S-DNA-lipid complex (lanes d and e) .
  • DNA was either uncoated (lanes b and d) or coated with rec A (lanes c and e) .
  • Figure 3A isolated DNA was amplified using primers CF8B/CF6 (top) and in Figure 3B using primers CF7B/CF6 (bottom) . Control samples were of DNA from
  • ⁇ CFTE/con (lane f) ; nonCF (N/N) lymphocytes (lane g) ; ⁇ F508 homozygote ( ⁇ / ⁇ F) lymphocytes (lane h) ; ⁇ F508 heterozygote
  • lymphocytes (lane i) ; and water (lane j) .
  • FIG. 4 shows allele-specific PCR analysis of CFPAC-1 cells transfected with rec A coated and uncoated 491 base fragments using the StarburstTM dendrimer-DNA complex to introduce the homologous DNA into the cells.
  • PCR analysis of CFPAC-1 DNA from control nontransfected (lane b) and mock-transfected without DNA (lanes c and d) Cells transfected with fragment 9 (lanes e and f) and control DNA from the N/N and N/ ⁇ F508 lymphocytes (lanes g and i) indicated the presence of wtCFTR sequences.
  • Figure 5 shows RT-PCR analysis of CFTR mRNA from HTE-11
  • the cDNA was amplified with primer CF17 (exon 9) and primers specific for normal (CF7C, 330-bp fragment) or mutant (CF8C, 327-bp fragment) sequences. Digestion with Xho I only showed the expected 221-bp and 106-bp restriction fragments for DNA amplified with CF17/CF8C. Nontransfected controls are in the C lanes. The marker (123-bp) is in lane M.
  • Figure 6 shows analysis of DNA amplified from first- strand CFTR cDNA from ⁇ CFTE29o- cells electroporated with rec A-coated 491-nucleotide fragments (lanes 6 and 7) or with rec A-coated or DNA fragments not coated with rec A, encapsulated as a gramicidin S-DNA-lipid complex (lanes 8 and 9) , respectively.
  • Cells were electroporated or transfected with the rec A-coated or uncoated 491 nucleotide fragments, and cytoplasmic RNA was isolated 7 days later.
  • CFTR mRNA was reverse-transcribed into first- strand CFTR cDNA.
  • the cDNA was amplified with CF17 (exon 9) primer and allele-specific primers for either normal (oligo N, 322-bp fragment) or mutant ( ⁇ F,321-bp fragment) sequences.
  • a 321-bp PCR fragment was produced in DNA from transfected ⁇ CFTE29o- cells when first-strand cDNA was amplified directly with the CF17/ ⁇ F primer pair in control cells (lane 4) .
  • control normal 16HBE14o- cells (lane 2) and electroporated and transfected cells (lanes 6-9) amplified with CF17/N produced a 322-bp band.
  • No DNA product was observed when cDNA from ⁇ CFTE/con cells was amplified with the CF17/N primers (lane 3) . Marker DNA (100-bp) is located in lanes 1, 5, and 10.
  • RNA from gramicidin S-DNA-lipid- transfected ⁇ CFTE29o- cells also contained wtCFTR mRNA whether or not the 491 nucleotide fragments were coated with rec A (lanes 8 and 9) .
  • Figure 7 is restriction enzyme digestion analysis of the allele-specific amplification product generated with primers CF1S/CF8C.
  • Primary nonCF airway epithelial cells were transfected with 488 base ssDNA fragments containing the ⁇ F508 mutation and an Xho I restriction enzyme site.
  • PCR amplification of the DNA by primers CF1B/CF7C gave a product of 389 bp.
  • fragments of 283 and 106 bp were detected (lanes 1 and 2) .
  • DNA was isolated from cells transfected with rec A-coated (lane 1) and uncoated (lane 2) 488 base fragments.
  • Figure 7 shows that using PCR analysis of the DNA from nonCF primary airway epithelial cells that the cells have undergone homologous replacement with a 488 nucleotide ⁇ F508 DNA fragment. Restriction enzyme analysis of the PCR product generated from the genomic exon 10 locus further shows that the exogenous fragment replaced the endogenous sequences and in this way confirmed that the gene therapy was successful.
  • Figure 8 shows PCR analysis of human ⁇ -globin DNA. Primers are listed in Table 3.
  • Figure 8 is a schematic representation of the RT-PCR and Dde I restriction -digestion analysis of 3-globin mRNA-derived cDNA.
  • Figure 9 shows fluorescent micrographs of allele- specific in si tu RT-PCR of two, one normal and one mutated, cell lines.
  • Figures 9A-D show allele-specific in si tu RT-PCR of a normal epithelial cell line (16HBE14o-) and a CF tracheal epithelial cell line homozygous for the ⁇ F508 mutation ( ⁇ CFTE290-) .
  • a primer pair specific for normal CFTR mRNA sequences (CF7B/CF22) seen in Tables 1 and 2 was used to assess wild-type CFTR expression in both cell lines.
  • Primer CF7B is allele-specific for wtCFTR and primer CF22 is non-allele-specific in exon 11.
  • the resultant amplification gives rise to a product that crosses intron- exon boundaries and therefore only amplifies mRNA-derived mutated CFTR cDNA.
  • Figure 9A is in si tu PCR of normal 16HBE14o-cells following DNase treatment and reverse transcription (+RT) .
  • area 51 seen in the colored original has varying blue green colors.
  • White areas 52 and 53 seen in the black and white Figure 9A are yellow in the color original .
  • Figure 9B is in si tu RT-PCR of mutated ⁇ CFTE29o- cells. As seen in Figure 9B, the wild-type CFTR DNA was not expressed in the mutated cells. On the colored photograph, there are no yellow areas or fluorescence observed. The Figure 9B is virtually all blue.
  • Figure 9C represents in si tu PCR of 16HBE140-
  • Figure 9D represents in si tu PCR of ⁇ CFTE29o- without reverse transcription (-RT) . Because there was no reverse transcription performed in the studies showing Figures 9C and 9D, there is no yellow fluorescence observed in either of these figures.
  • Figures 9B-D show some very slight green but the light areas are mostly blue seen as black in
  • Figures 9B-D Only Figure 9A is positive for the presence of tCFTR mRNA.
  • Figure 10 is a color fluorescent photograph of cells submitted to human allele-specific in si tu RT-PCR of a section normal bronchial epithelial tissue. Mucous (M) cells and serous (S) cells are indicated. In Figure 10, the light areas 62 and 63 which are yellow in the original are for serous cells, and areas 61 are blue green for mucous.
  • RT-PCR was carried out as described above for Figures 9A-D. Fluorescence was observed primarily in serous cells and only to a smaller degree in the mucous cells. Results indicate the presence of wtCFTR mRNA predominantly in serous cells and not in mucous cells.
  • the method of detection employs polymerase chain reaction (PCR) amplification of mRNA-derived cDNA.
  • the primers used in are allele-specific and will differentiate between the endogenous mutant ( ⁇ F508) cystic fibrosis transmembrane conductance regulator (CFTR) genes and therapeutic wild-type CFTR DNA.
  • One primer is non-allele- specific and in an exon adjacent to the exon containing the mutation and one primer is specific for either ⁇ F508 or wild-type CFTR. Effectively 3 primers are necessary for this analysis.
  • FIG. 11-13 The studies represent analysis of the expression of specific CFTR alleles ( ⁇ F508 and wild-type) in cells and tissue sections. Expression of CFTR in cells was determined using PCR primers that differentiate between
  • Figure 11 is allele-specific in si tu RT-PCR in human tissue showing assessment of wtCFTR in sections of human airway epithelial tissue.
  • Figure IIA shows wtCFTR mRNA detected in normal bronchial tissue with primers CF17/CF7C (upper panel) , but not with the ⁇ F508 primers CF17/CF8C (lower panel) .
  • the yellow fluorescence is visible over the whole photograph in upper panel .
  • Lower panel shows only traces of the fluorescence, seen as small white spots. Wild-type CFTR was primarily expressed and the expression detected in the submucosal glands in serous cells (S) and not in mucous cells (M) .
  • Figure 11B shows wtCFTR mRNA detected in normal human colon tissue with allele-specific primers CF17/CF7C for wt DNA (left top panel) , but not with allele-specific primers
  • CF17/CF8C to mutation top right panel
  • Conventional in si tu hybridization has shown CFTR mRNA in this region.
  • the crypt shows the strongest signal as seen in the bottom panel.
  • Figure 12 is allele-specific in si tu RT- PCR in human bronchial epithelial cell line. Analysis of cultured airway epielial cells shows that the primers were able to differentiate between the expression of wild-type and ⁇ F508 CFTR ( Figures 12A and 12B) .
  • Figure 12A shows wild-type (wt) CFTR mRNA detected in normal bronchial epithelial cells (16HBE14o-) using a wt- specific oligonucleotide primer pair (CF17/CF7C) , top panel. As seen in Figure 12A bottom panel, no signal was detected with a ⁇ F508-specific oligonucleotide primer pair - (CF17/CF8C) .
  • Figure 12B shows ⁇ F508 CFTR mRNA detected in CF bronchial epithelial cells (CFBE410-) that were homozygous for the ⁇ F508 mutation when the ⁇ F508-specific primer pair was used, ( Figure 15B, bottom panel) . No signal was detected when primers specific for the wt-allele were used ( Figure 12B, top panel) .
  • Figure 12C shows a mixed population of 16HBE140- and ⁇ CFBE41o- cells where it was possible to identify those cells expressing the wtCFTR allele with primers CF17/CF7C.
  • the left panel was with reverse transcription (+RT) where some green fluorescence is visible.
  • the right panel which shows samples which were not reverse transcribed, does not show visible fluorescence (-RT) .
  • mice Normal mice were transfected with liposome-DNA complexes by intralung instillation. DNA fragments were comprised of mouse CFTR (mCFTR) exon 10 and flanking intron 9 and 10 DNA sequences. The entire fragment was 783-bp and contained a ⁇ F508 mutation (a TTT deletion of codon 508) and a silent mutation (T>C) to give rise to a unique Kpn I restriction enzyme cleavage site.
  • the expression of this exogenous sequence was assayed as RNA from tissue harvested 4 days after the mice were transfected. Tissue was harvested from the trachea, lungs and the liver. Extraction of the RNA occurred immediately after the tissue was removed from the animal . The RNA was then reverse transcribed and first strand cDNA was amplified by allele- specific PCR with primers mCF12r and mCF ⁇ F508-3. Results are seen in Figure 13.
  • Figure 13 is allele-specific RT-PCR analysis of RNA samples isolated from transfected mice at day 4 post- transfection.
  • Figure 13A lanes 1-3 show lung, lanes 4-6 show trachea, lanes 7-9 show heart tissue. Lanes 1, 4, and 7 were transfected with 4 ⁇ g of DNA. Lanes 2, 5 and 8 were transfected with 20 ⁇ g DNA. Lanes 3, 6 and 9 were controls without DNA transfection. Lanes 10 (liver) and 11 (lung)
  • Lane 12 is water control.
  • the molecular weight marker is a 123-bp ladder.
  • Figure 13B shows Kpn 1 restriction enzyme cleavage of a second round PCR amplification product from lanes 1 and
  • Lane 13A shows lung sample from mice transfected with 4 ⁇ g DNA without Kpn 1 digestion. Lanes 2 and 3 Kpn 1 show digest of PCR products from mice transfected with 4 and 20 ⁇ g, respectively. Lane 4 shows nontransfected control amplification and Kpn 1 digest. Lane 5 shows Kpn 1 digest of PCR product from a ⁇ F508 heterozygote control mouse; and lane 6, shows water control.
  • the marker (lane M) is a 1-kb molecular weight marker (Gibco BRL) .
  • the results of the initial amplification indicated that there was, in fact, ⁇ F508 CFTR mRNA expressed in the lung tissue ( Figure 13A) .
  • the assay of the invention is used to quantify the number of cells within a population of CF cells transfected in vi tro or in vivo expressing wt CFTR.
  • the method of the invention is applied to the alteration of the genetic defects associated with CF disease.
  • the method is suitable and applicable to cells with other genetic defects for which the wild-type or otherwise normal DNA sequence is known.
  • Also within this invention is the detection of alterations of DNA sequences associated with genetic diseases in animals other than humans. These detectable genetic diseases can either be induced as in the case of transgenic animals, or they can be corrected as in the case of gene therapy.
  • the current invention is useful for verification and assessment of gene therapy used for correction of genetic disorders. While the invention was proven to work for cystic fibrosis, almost all genetic diseases can be detected according to the invention.
  • this assay and a method also provides a means for verification of altering DNA sequences which do not express a gene product, including alterations in regulatory sequences, intron sequences, and the substitution of redundant codon sequences.
  • EXAMPLE 1 Preparation of the Wild-type 491 Base Pair DNA and Primers This example illustrates preparation of the wt 491 bp DNA and primers .
  • the 491 bp fragment was generated using the T6/20
  • the amplified fragment was analyzed on a 1% agarose gel, and then amplified in bulk in 20 separate PCR amplifications each containing 50 ng.
  • the 491 bp fragments were purified by phenol :chloroform:isoamyl alcohol
  • PCR conditions for individual primers are as follows :
  • Cycle denaturation 94°C for 30 sec, annealing 49°C for 20 sec extension 72°C for 20 sec with a 4 sec/cycle increase in extension time for 35 cycles.
  • Cycle denaturation 95°C for 60 s, annealing 56°C for 60 s, 72°C for 120 s; 35 cycles with an 8 min extension on the last cycle; and Mg +2 mM.
  • Primers 0.5 ⁇ M;DNA, 50-100 ng;
  • Cycle denaturation 95°C for 60 s, annealing 59°C for 60 s, 72°C for 90 s; 35 cycles with an 8 min extension on the last cycle; and Mg +2 2 mM.
  • CF17/CF7C-8C 330-bp (N) AND 327-bp ( ⁇ F) , Primers, 1 ⁇ M;
  • Cycle denaturation 94°C for 20 s; annealing, 58°C for 60 s; extension, 72°C for 60 s for 35 cycles with a with a 5 min extension on the last cycle; and Mg +2 , 2.5 mM.
  • CF17/CF22 470-bp ( ⁇ F) and 473-bp (N) fragments. Conditions were as follows:
  • Cycle denaturation 94°C for 20 s, annealing 58°C for 20 s, 72°C for 30 s for 35 cycles.
  • mCFN-3/mCFllR Cycle: denaturation 94°C for 20 s, annealing 58°C for 20 s, 72°C for 30 s, for 35 cycles.
  • EXAMPLE 3 Hybridization of Probes to Filters Extrapolate to in si tu Conditions This example illustrates the conditions for hybridization of probes to filters to extrapolate to in si tu conditions.
  • DNA fragments were separated by agarose gel electrophoresis. The gels with the fragments were incubated in 0.4 N NaOH containing 0.6 M NaCl for 30 min to denature DNA and then washed one time with 1.5 M NaCl, 0.5 M Tris-HCl for 30 min.
  • the DNA was transferred to a Gene Screen Plus membrane
  • Tris-HCl Tris-HCl.
  • the membranes were prehybridized for 1 hour at 37°C in 6 X SSC, 5 X Denhardt's, 1% SDS, and 100 ⁇ g/ml of denatured salmon sperm DNA.
  • oligonucleotide probes (oligo N or oligo ⁇ F; 10 ng) were radiolabelled by reaction with 20 units of T4 kinase and 40 ⁇ Ci 32 P- ⁇ -ATP for 30 min at 37°C. Unincorporated nucleotides were removed by centrifugation of the reaction mix through a minispin column.
  • This example illustrates reverse transcriptase polymerase chain reaction procedure used in detection of SFHR in cystic fibrosis.
  • the PCR reaction was performed again using Perkin Elmer solutions containing a 50 ⁇ l final volume: IX PCR buffer II, 700 ⁇ M each nucleotide, 10 uM each primer, 4.5mM MgCl 2 , 70 ⁇ M fluorescently labelled d-UTP, and 10 units of Taq (is) DNA polymerase.
  • a hotstart was performed with the in situ PCR system 1000, thermal cycler, 25 cycles, target - temperature, 59°C.
  • This example describes RT-PCR procedure used for detection of DNA expressions in various tissues.
  • Tissues either lung, bronchial or colon cells, were fixed for 4 h in paranormal-dehyde fixative PFA followed by incubation of 30% sucrose in PBS overnight. Tissue sections were frozen in Octanol using Freon and liquid nitrogen. Slides were then stored at -70° C until used. Before use, slides were placed in a 55°C oven for 10-30 minutes to help bind the cells tightly to the slides. Cells and tissue were then digested with pepsin (2mg/ml) , for varying periods of time (15-45 min) that were dependent on the fixation time. The slides were washed 1 X with 1 X PBS and 70% ethanol and then air dried.
  • the PCR reaction was carried out in a 50 ⁇ l volume with
  • PCR amplification was performed following a hot start (70°C for 5 - 10 min, then 94°C for 2 min) with the Perkin Elmer in si tu PCR System 1000 Therma Cycler.
  • the Cycle conditions were: 94°C/60 s, denaturation; 59°C/2 min, annealing; 70°C/60 s, extension for 20 cycles.
  • This example describes the assay used for differentiation between gene expression in tested tissue or cells and gene expression in the normal wild-type tissue or cells .
  • the assay comprises the following steps: (a) obtaining tissue samples as follows:
  • step (b) fixing, digesting and reverse transcribing both samples (1) and (2) of step (a) using procedures described in Examples 4 and 5, on separate slides, each slide containing three drops or sections of the same tissue or cells to ensure the correct interpretation, each drop to be treated as follows:
  • one drop is submitted to DNase digestion to expose single strand mRNA to be used as a template for production of cDNA and also to eliminate tissue or cells own DNA and prevent false positive results from this DNA, and subsequently this drop is submitted to reverse transcription to produce first strand cDNA from the - mRNA template;
  • step (c) amplifying the cDNA of step (b) using polymerase chain reaction in the presence of the allele-specific primers for wild-type DNA or tested experimental and/or mutated DNA, depending on what is being tested, under conditions and in a solution comprising all necessary nucleotides to obtain the cDNA in sufficient quantity for assay, wherein at least one nucleotide in the solution or in the primer is labelled with a non-interfering radioactive, immunochemical, fluorescent or other labeled marker detectable by spectroscopic, autoradiographic, immunocytochemical or enzymatic detection means;
  • step (e) detecting the presence of the labeled marker in the amplified product which detects either the mutated DNA or the wild-type DNA, depending on the allele-specific primers used for PCR amplification, by detecting the presence and quantity of expression of mutated or normal wild-type DNA in the tested sample and/or in the control sample; and (f) comparing qualitatively and quantitatively the results obtained in step (e) , for tested and control samples wherein the presence of the labeled nucleotide in the amplified product using wild-type allele-specific primers determines the expression of the normal nonmutated DNA and wherein the absence of the labeled nucleotide in the amplified product using mutated allele-specific primers determines the absence of the expression of the mutated allele, and wherein the amount or the level of the labeled nucleotide can be quantified and the number of expressions of the mutated allele vis-a-vis expressions of the wild ⁇ type DNA determined by, for example using NIH
  • the Assay for Detection of Various Genetic Diseases and for Qualitative and Quantitative Assessment of the Gene Therapy Success This example illustrates an assay used for detection of various genetic diseases and for qualitative and quantitative assessment of the gene therapy success or for designing the gene therapy protocol.
  • Genetic diseases for which this assay is suitable are selected from the group consisting of cystic fibrosis, Fanconi's anemia, sickle cell anemia, retinitis pigmentosa, xeroderma pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, Duchenne' s muscular dystrophy, and Tay-Sachs disease.
  • the endogenous tissue or cells are obtained from individuals suffering from, suspected to be suffering from or having the family history of cystic fibrosis or from the individual subjected to gene therapy by replacing the cystic fibrosis transmembrane conductive regulator (CFTR) gene with wild ⁇ type nonmutated DNA.
  • CFTR cystic fibrosis transmembrane conductive regulator
  • the endogenous tissue or cells are obtained from individual suffering from sickle cell anemia.
  • the endogenous tissue or cells are obtained from individual suffering from xeroderma pigmentosa. In the assay described in Example 6, the endogenous tissue or cells are obtained from individual suffering from genetic disease listed above.
  • EXAMPLE 8 The Assay for Detection of Precancerous Cells and for Qualitative and Quantitative Assessment of the Gene
  • This example illustrates an assay used for detection of - precancerous conditions in the cells having the normal phenotype .
  • the assay as described in Example 6 is used for detection of cancerous mutations in the cells as well as for designing the suitable protocol for gene therapy by replacing the mutated DNA within the cells with nonmutated DNA and for verification that such replacement did, in fact happened.
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  • MOLECULE TYPE synthetic oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO 51:
  • MOLECULE TYPE synthetic oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO 52:
  • MOLECULE TYPE synthetic oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO 53:
  • MOLECULE TYPE synthetic oli onucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO 54: ATAGGAAACA CCAAAGATGA 20
  • MOLECULE TYPE synthetic oligonucleotide
  • SEQUENCE DESCRIPTION SEQ ID NO 55:

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Abstract

On décrit un procédé et un dosage à amplification PCR in situ à transcriptase inverse spécifique d'allèles permettant une détection et une différentiation entre l'expression de séquences non mutées d'ADN de type sauvage et de séquences endogènes mutées d'ADN, in vitro ou in vivo. Ce procédé sert à la vérification, et à l'évaluation et à la surveillance diagnostiques d'une thérapie génique reposant sur le remplacement homologue thérapeutique de petits fragments, à l'évaluation et à la surveillance diagnostiques de thérapies géniques à base d'ADN complémentaire, à l'analyse de l'expression génique d'allèles spécifiques pendant le développement foetal, et à l'évaluation diagnostique de l'expression d'allèles impliqués dans des mutations cancéreuses.
PCT/US1996/016162 1995-10-10 1996-10-08 Detection de mutations specifiques d'alleles par amplification pcr in situ a transcriptase inverse, specifique d'alleles WO1997013869A1 (fr)

Priority Applications (1)

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AU72626/96A AU7262696A (en) 1995-10-10 1996-10-08 Detection of allele-specific mutations by allele-specific in situ reverse transcriptase polymerase chain reaction

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US525495P 1995-10-10 1995-10-10
US60/005,254 1995-10-10

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725422A (zh) * 2021-02-26 2021-04-30 山东康华生物医疗科技股份有限公司 一种用于hras g13r突变检测的引物、探针以及试剂盒

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5364790A (en) * 1993-02-16 1994-11-15 The Perkin-Elmer Corporation In situ PCR amplification system
US5449605A (en) * 1988-10-14 1995-09-12 Georgetown University Method of detecting a predisposition to cancer by detecting a deletion polymorphism in the gene for human poly (ADP-ribose) polymerase
US5478724A (en) * 1991-08-16 1995-12-26 The Rockefeller University Lentivirus-specific nucleotide probes and methods of use
US5480783A (en) * 1994-03-31 1996-01-02 The Perkin-Elmer Corporation Method for reducing background signals in DNA replication/detection assays
US5538871A (en) * 1991-07-23 1996-07-23 Hoffmann-La Roche Inc. In situ polymerase chain reaction
US5567586A (en) * 1995-05-18 1996-10-22 Thomas Jefferson University Methods of indentifying solid tumors with chromosome abnormalities in the ALL-1 region
US5576178A (en) * 1991-10-04 1996-11-19 The Childrens Hospital Of Philadelphia Method of detecting genetic deletions and mutations associated with DiGeorge syndrome, Velocardiofacial syndrome, charge association, conotruncal cardiac defect, and cleft palate and probes useful therefor

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5449605A (en) * 1988-10-14 1995-09-12 Georgetown University Method of detecting a predisposition to cancer by detecting a deletion polymorphism in the gene for human poly (ADP-ribose) polymerase
US5538871A (en) * 1991-07-23 1996-07-23 Hoffmann-La Roche Inc. In situ polymerase chain reaction
US5478724A (en) * 1991-08-16 1995-12-26 The Rockefeller University Lentivirus-specific nucleotide probes and methods of use
US5576178A (en) * 1991-10-04 1996-11-19 The Childrens Hospital Of Philadelphia Method of detecting genetic deletions and mutations associated with DiGeorge syndrome, Velocardiofacial syndrome, charge association, conotruncal cardiac defect, and cleft palate and probes useful therefor
US5364790A (en) * 1993-02-16 1994-11-15 The Perkin-Elmer Corporation In situ PCR amplification system
US5527510A (en) * 1993-02-16 1996-06-18 The Perkin-Elmer Corporation In situ PCR amplification system
US5480783A (en) * 1994-03-31 1996-01-02 The Perkin-Elmer Corporation Method for reducing background signals in DNA replication/detection assays
US5567586A (en) * 1995-05-18 1996-10-22 Thomas Jefferson University Methods of indentifying solid tumors with chromosome abnormalities in the ALL-1 region

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NUCLEIC ACIDS RESEARCH, Volume 22, Number 15, issued 11 August 1994, HOFFMAN-LAROCHE, "Research Genetics". *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725422A (zh) * 2021-02-26 2021-04-30 山东康华生物医疗科技股份有限公司 一种用于hras g13r突变检测的引物、探针以及试剂盒

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