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  • C12Q2539/00—Reactions characterised by analysis of gene expression or genome comparison
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  • C12Q2539/105—Involving introns, exons, or splice junctions

Definitions

• This invention relates to a gene which has been associated with breast cancer where the gene is found to be mutated. More specifically, this invention relates to five unique coding sequences of BRCA2 gene
• BRCA2 (om ⁇ 1) , BRCA2 (om ⁇ 2) , BRCA2 (om ⁇ 3) , BRCA2 (om ⁇ 4) , and BRCA2 (om,5) identified in human subjects which define five novel haplotypes.
• Locating one or more mutations in the BRCA2 region of chromosome 13 provides a promising approach to reducing the high incidence and mortality associated with breast cancer through the early detection of women and men at high risk. These individuals, once identified, can be targeted for more aggressive prevention programs. Screening is carried out by a variety of methods which include karyotyping, probe binding and DNA sequencing.
• DNA sequencing technology genomic DNA is extracted from whole blood and the coding regions of the BRCA2 gene are amplified. Each of the coding regions may be sequenced completely and the results are compared to the normal DNA sequence of the gene. Alternatively, the coding sequence of the sample gene may be compared to a panel of known mutations or other screening procedure before completely sequencing the gene and comparing it to a normal sequence of the gene.
• the BRCA2 gene is divided into 27 separate exons. Exon 1 is noncoding, in that it is not part of the final functional BRCA2 protein product.
• the BRCA2 coding region spans roughly 10433 base pairs (bp) over 70 kb. Each exon consists of 100-600 bp, except for exons 10, 11 and 27. The full length mRNA is 11-12 kb.
• each exon is amplified separately and the resulting PCR products are sequenced in the forward and reverse directions.
• exons 10, 11 , and 27 are so large, we have divided them into three, twenty-one, and two overlapping PCR fragments (respectively) of approximately 250-625 bp each (segments "A” through “C” of exon 10, "A” through “U” of exon 11 , and "A” through “B” of exon 27).
• BRCA2 Breast Cancer Information Core
• GenBank Accession Number U43746
• BIC Breast Information Core
• the present invention is based on the discovery of the correct genomic BRCA2 sequence and five novel sequence haplotypes found in normal human subjects of the BRCA2 gene. It is an object of this invention to provide the correct intronic/exonic sequence of the BRCA2 gene.
• a person skilled in the art of genetic susceptibility testing will find the present invention useful for: a) identifying individuals having a normal BRCA2 gene with no coding sequence mutations, who therefore cannot be said to have an increased genetic susceptibility to breast or ovarian cancer from their BRCA2 genes; b) avoiding misinterpretation of normal polymorphisms found in the BRCA2 gene; c) determining the presence of a previously unknown mutation in the BRCA2 gene; d) identifying a mutation in exon 11 of BRCA2 which indicates a predisposition or higher susceptibility to ovarian cancer than breast cancer (i.e., resides in the putative "ovarian cancer cluster" region); e) probing a human sample of the BRCA2 gene by allele to determine the presence or absence of either polymorphic alleles or mutations; f) performing gene therapy with the correct BRCA2 gene sequence.
• FIGURE 1 shows the GenBank genomic sequence of BRCA2 (Accession Number U43746).
• the lower case letters denote intronic sequences and the upper case letters denote exonic sequences. Incorrect exonic sequences at exons 5 and 16 are shown with boldface type.
• FIGURE 2 shows the corrected genomic sequence of BRCA2.
• the lower case letters denote intronic sequences and the upper case letters denote exonic sequences. Corrected intronic and exonic sequences at exons 5, 11 and 15 are shown with boldface type.
• FIGURE 3 shows the alternative alleles at polymorphic sites along a chromosome which can be represented as a unit or "haplotype" within a gene such as BRCA2.
• the haplotype that is in GenBank (GB) is shown with light shading.
• Five additional haplotypes are shown in FIGURE 3 (encompassing the alternative alleles found at nucleotide sites 1093, 1342, 1593, 2457, 2908, 3199, 3624, 4035, 7470 and 9079).
• - 5) are represented with mixed dark and light shading (numbers 2, 4, 6, 8 and 10 from left to right). In total, 5 of 10 haplotypes along the BRCA2 gene are unique.
• Breast and Ovarian cancer is understood by those skilled in the art to include breast, ovarian and pancreatic cancer in women and also breast, prostate and pancreatic cancer in men.
• BRCA2 is associated with genetic susceptibility to breast, ovarian and pancreatic cancer. Therefore, claims in this document which recite breast and/or ovarian cancer refer to breast, ovarian, prostate, and pancreatic cancers in men and women.
• Coding sequence refers to those portions of a gene which, taken together, code for a peptide (protein), or which nucleic acid itself has function.
• Protein or “peptide” refers to a sequence of amino acids which has function.
• BRCA2 (omi) refers to the genomic BRCA2 sequence disclosed in Genbank (Accession Number U43746) wherein, (1 ) a 10 bp stretch (5'-TTTATTTTAG-3') is intronic at 3' end of intron 4, rather than at the 5' end of exon 5; and
• BRCA2 (omi 1 5)
• introns particularly the slice sites adjacent to the exons.
• sequences were found by end to end sequencing of the BRCA2 gene from 5 individuals randomly drawn from the population and who were documented to have no family history of breast or ovarian cancer.
• the sequenced exons were found not to contain any truncating mutations.
• the change of a nucleic acid at a polymorphic site lead to a codon change and a change of amino acid from the previously published standard in GenBank (see TABLE III).
• the frequency of occurrence of a nucleic acid change was found to differ from the published frequency or was newly determined.
• Normal DNA sequence also called “normal gene sequence” refers to a nucleic acid sequence, the nucleic acid of which are known to occur at their respective positions with high frequency in a population of individuals who carry the gene which codes for a normally functioning protein, or which itself has normal function.
• Normal Protein Sequence refers to the protein sequence, the amino acids of which are known to occur with high frequency in a population of individuals who carry the gene which codes for a normally functioning protein.
• Normal Sequence refers to the nucleic acid or protein sequence, the nucleic or amino acids of which are known to occur with high frequency in a population of individuals who carry the gene which codes for a normally functioning protein, or which nucleic acid itself has a normal function.
• Haplotype refers to a series of specific alleles within a gene along a chromosome.
• “Functional allele profile” refers a list of those alleles in the normal population which have the funll function. “Mutation” refers to a base change or a gain or loss of base pair(s) in a
• DNA sequence which results in a DNA sequence coding for a non-functional protein or a protein with substantially reduced or altered function.
• Polymorphism refers to a base change in a DNA sequence which is not associated with known pathology.
• Primary refers to a sequence comprising about 15 or more nucleotides having a sequence complementary to the BRCA2 gene. Other primers which can be used for primer hybridization will be known or readily ascertainable to those skilled in the art.
• Substantially complementary to refers to primer sequences which hybridize to the sequences provided under stringent conditions and/or sequences having sufficient homology with BRCA2 sequences, such that the allele specific oligonucleotide primers hybridize to the BRCA2 sequences to which they are complimentary.
• isolated nucleic acids refers to nucleic acids substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which they may be associated. Such association is typically either in cellular material or in a synthesis medium.
• Bio sample or “body sample” refers to a sample containing DNA oatained from a biological source.
• the sample may be from a living, dead or even archeological source from a variety of tissues and cells.
• body fluid e.g. blood (leukocytes), urine (epithelial cells), saliva, breast milk, menstrual flow, cervical and vaginal secretions, etc.
• body fluid e.g. blood (leukocytes), urine (epithelial cells), saliva, breast milk, menstrual flow, cervical and vaginal secretions, etc.
• skin e.g. hair roots/follicle, mucus membrane (e.g. buccal or tongue cell scrapings), cervicovaginal cells (from PAP smear, etc.), lymphatic tissue, internal tissue (normal or tumor).
• Vector refers to any polynucleotide which is capable of self replication or inducing integration into a self-replicating polynucleotide. Examples include polynucleotides containing an origin or replication or an integration site. Vectors may be intergrated into the host cell's chromosome or form an autonomously replicating unit.
• a tumor growth inhibitor refers to a molecule such as, all or a fragment of BRCA2 protein, a BRCA2 polypeptide, or a functional equivalent 5 thereof that is effective for preventing the formation of, reducing, or eliminating a transformed or malignant phenotype of breast or ovarian cancer cells.
• a BRCA2 polypeptide refers to a BRCA2 polypeptide either directly derived from the BRCA2 protein, or homologous to the BRCA2 protein, or a o fusion protein consisting of all or fragments of the BRCA2 protein and polypeptides.
• a functional equivalent refers to a molecule including an unnatural BRCA2 polypeptide, a drug or a natural product which retains substantial biological activity as the native BRCA2 protein.
• the activity and function of 5 BRCA2 protein may include transactivation, granin, DNA repair, among others.
• a target polynucleotide refers to the nucleic acid sequence of interest, for example, the BRCA2 encoding polynucleotide.
• Other primers which can be used for primer hybridization will be known or readily o ascertainable to those of skill in the art.
• the invention in several of its embodiments includes: an isolated DNA sequence of the BRCA2 coding sequence as set forth in SEQ ID NO:4, 6, 8, 10, and 12, a protein sequence of the BRCA2 protein as set forth in SEQ ID NO:5, 7, 9, 11 , 13, a method of identifying individuals having a normal 5 BRCA2 gene with no increased risk for breast and ovarian cancer, a method of detecting an increased genetic susceptibility to breast and ovarian cancer in an individual resulting from the presence of a mutation in the BRCA2 coding sequence, a method of performing gene therapy to prevent or treat a tumor, a method of protein replacement therapy to prevent or treat a tumor, a o diagnostic reagent comprising all or fragments of the disclosed BRCA2 cDNA and protein sequences.
• nucleic acid specimen in purified or non-purified form, can be utilized as the starting nucleic acid, providing it contains, or is suspected of containing, the specific nucleic acid sequence containing a polymorphic or a mutant allele.
• the process may amplify, for example, DNA or RNA, including mRNA and cDNA, wherein DNA or RNA may be single stranded or double stranded.
• RNA is to be used as a template
• enzymes and/or conditions optimal for reverse transcribing the template to DNA would be utilized.
• a DNA-RNA hybrid which contains one strand of each may be utilized.
• a mixture of nucleic acids may also be employed, or the nucleic acids produced in a previous method such as an amplification reaction using the same or different primers may be so utilized.
• the specific nucleic acid sequence to be amplified i.e., the polymorphic and/or the mutant allele, may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid.
• a variety of amplification techniques may be used such as ligating the DNA sample or fragments thereof to a vector capable of replication or incorporation into a replicating system thereby increasing the number of copies of DNA suspected of containing at least a portion of the BRCA2 gene.
• Amplification techniques include so called "shot gun cloning". It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as contained in whole human DNA.
• DNA utilized herein may be extracted from a body sample, such as blood, tissue material and other biological sample by a variety of techniques such as that described by Maniatis, et al. in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, p 280-281 , 1982). If the extracted sample is impure, it may be treated before amplification with an amount of a reagent effective to open the cells, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily.
• the isolated DNA may be cleaved into fragments by a restriction endonuclease or by shearing by passing the DNA containing mixture through a 25 gauge needle from a syringe to prepare 1- 1.5 kb fragments.
• the fragments are then ligated to a cleaved vector (virus, plasmid, transposon, cosmid etc.) and then the recombinant vector so formed is then replicated in a manner typical for that vector.
• the deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90°-100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein "agent for polymerization”), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization may also be added together with the other reagents if it is heat stable.
• This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions.
• the temperature is generally no greater than about 40°C. Most conveniently the reaction occurs at room temperature.
• thermostable DNA polymerase such as Taq, higher temperature may be used.
• the allele specific oligonucleotide primers are useful in determining whether a subject is at risk of having breast or ovarian cancer, and also useful for characterizing a tumor. Primers direct amplification of a target polynucleotide prior to sequencing. These unique BRCA2 oligonucleotide primers for exons 2-27 shown in TABLE II were designed and produced specifically to optimize amplification of portions of BRCA2 which are to be sequenced.
• the primers used to carry out this invention embrace oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization.
• Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH.
• the primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition.
• the oligonucleotide primer typically contains 18-28 bp plus in some cases an M13 "tail" for convenience.
• Primers used to carry out this invention are designed to be substantially complementary to each strand of the genomic locus to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' sequences flanking the mutation to hybridize therewith and permit amplification of the genomic locus. Oligonucleotide primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of polymorphic locus relative to the number of reaction steps involved. Typically, one primer is complementary to the negative (-) strand of the polymorphic locus and the other is complementary to the positive (+) strand.
• the product of the chain reaction is a discreet nucleic acid duplex with termini corresponding to the ends of the specific primers employed.
• oligonucleotide primers of the invention may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof.
• diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al., Tetrahedron Letters, 22:1859-1862, 1981.
• One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.
• the agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes.
• Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase, polymerase muteins, reverse transcriptase, other enzymes, including heat- stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation), such as Taq polymerase.
• Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each polymorphic locus nucleic acid strand.
• the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths.
• the newly synthesized strand and its complementary nucleic acid strand will form a double-stranded molecule under hybridizing conditions described above and this hybrid is used in subsequent steps of the process.
• the newly synthesized double-stranded molecule is subjected to denaturing conditions using any of the procedures described above to provide single-stranded molecules.
• the steps of denaturing, annealing, and extension product synthesis can be repeated as often as needed to amplify the target polymorphic locus nucleic acid sequence to the extent necessary for detection.
• the amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion. Amplification is described in PCR. A Practical Approach, ILR Press, Eds. M. J. McPherson, P. Quirke, and G. R. Taylor, 1992.
• the amplification products may be detected by Southern blots analysis, without using radioactive probes.
• a small sample of DNA containing a very low level of the nucleic acid sequence of the polymorphic locus is amplified, and analyzed via a Southern blotting technique or similarly, using dot blot analysis.
• the use of non- radioactive probes or labels is facilitated by the high level of the amplified signal.
• probes used to detect the amplified products can be directly or indirectly detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.
• Sequences amplified by the methods of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, etal., Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad. Sci. U.S.A., 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landgren, et al., Science, 241 :1007. 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landgren, et al., Science, 242:229-237, 1988).
• the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art.
• Alternative methods of amplification have been described and can also be employed as long as the BRCA2 locus amplified by PCR using primers of the invention is similarly amplified by the alternative means.
• Such alternative amplification systems include but are not limited to self-sustained sequence replication, which begins with a short sequence of RNA of interest and a T7 promoter. Reverse transcriptase copies the RNA into cDNA and degrades the RNA, followed by reverse transcriptase polymerizing a second strand of DNA.
• nucleic acid sequence-based amplification is nucleic acid sequence-based amplification (NASBA) which uses reverse transcription and T7 RNA polymerase and incorporates two primers to target its cycling scheme.
• NASBA can begin with either DNA or RNA and finish with either, and amplifies to 10° * copies within 60 to 90 minutes.
• nucleic acid can be amplified by ligation activated transcription (LAT).
• LAT ligation activated transcription
• Amplification is initiated by ligating a cDNA to the promoter oligonucleotide and within a few hours, and amplification is 10 ⁇ to 10 9 fold.
• the Q ⁇ replicase system can be utilized by attaching an RNA sequence called MDV-1 to RNA complementary to a DNA sequence of interest. Upon mixing with a sample, the hybrid RNA finds its complement among the specimen's mRNAs and binds, activating the replicase to copy the tag-along sequence of interest.
• Another nucleic acid amplification technique ligase chain reaction (LCR), works by using two differently labeled halves of a sequence of interest which are covalently bonded by ligase in the presence of the contiguous sequence in a sample, forming a new target.
• the repair chain reaction (RCR) nucleic acid amplification technique uses two complementary and target-specific oligonucleotide probe pairs, thermostable polymerase and ligase, and DNA nucleotides to geometrically amplify targeted sequences.
• a 2-base gap separates the oligonucleotide probe pairs, and the RCR fills and joins the gap, mimicking normal DNA repair.
• Nucleic acid amplification by strand displacement activation (SDA) utilizes a short primer containing a recognition site for hincll with short overhang on the 5' end which binds to target DNA.
• a DNA polymerase fills in the part of the primer opposite the overhang with sulfur-containing adenine analogs.
• Hincll is added but only cuts the unmodified DNA strand.
• a DNA polymerase that lacks 5' exonuclease activity enters at the site of the nick and begins to polymerize, displacing the initial primer strand downstream and building a new one which serves as more primer.
• SDA produces greater than 10 7 -fold amplification in 2 hours at 37°C. Unlike PCR and LCR, SDA does not require instrumented Temperature cycling.
• Another method is a process for amplifying nucleic acid sequences 5 from a DNA or RNA template which may be purified or may exist in a mixture of nucleic acids.
• the resulting nucleic acid sequences may be exact copies of the template, or may be modified.
• the process has advantages over PCR in that it increases the fidelity of copying a specific nucleic acid sequence, and it allows one to more efficiently detect a particular point mutation in a 0 single assay.
• a target nucleic acid is amplified enzymatically while avoiding strand displacement. Three primers are used.
• a first primer is complementary to the first end of the target.
• a second primer is complementary to the second end of the target.
• a third primer which is similar to the first end of the target and which is substantially complementary 5 to at least a portion of the first primer such that when the third primer is hybridized to the first primer, the position of the third primer complementary to the base at the 5' end of the first primer contains a modification which substantially avoids strand displacement.
• Sample DNA or RNA may be amplified by PCR, labeled with a fluorescent tag, and hybridized to the microarray. Examples of this technology are provided in U.S. Patents 5,510, 270, U.S. 5,547,839, incorporated herein by reference. o All exonic and adjacent intronic sequences of the BRCA2 gene were obtained by end to end sequencing of five normal subjects in the manner described above followed by analysis of the data obtained. The data obtained provided us with the opportunity to establish the correct intronic/exonic structure of the BRCA2 gene.
• polynucleotide(s) which result from either sense or antisense transcription of any exon or the entire coding sequence or fragments of BRCA2 gene may be used for gene therapy.
• a variety of methods are known for gene transfer, any of which might be available for use.
• DNA conjugated to a target receptor structure such as a diptheria toxin, an antibody or other suitable receptor.
• a target receptor structure such as a diptheria toxin, an antibody or other suitable receptor.
• Direct injection by particle bombardment for example, the
• DNA may be coated onto gold particles and shot into the cells.
• Receptor-Mediated Gene Transfer DNA is linked to a targeting molecule that will bind to specific cell- surface receptors, inducing endocytosis and transfer of the DNA into mammalian cells.
• a targeting molecule that will bind to specific cell- surface receptors, inducing endocytosis and transfer of the DNA into mammalian cells.
• One such technique uses poly-L-lysine to link asialoglycoprotein to DNA.
• An adenovirus is also added to the complex to disrupt the lysosomes and thus allow the DNA to avoid degradation and move to the nucleus. Infusion of these particles intravenously has resulted in gene transfer into hepatocytes.
• MoMLV Moloney Murine Leukemia Virus
• AAV Adeno-Associated Virus
• HSV herpes simplex virus
• HSV herpes simplex virus
• HIV human immunodeficiency virus
• GENE REPLACEMENT AND REPAIR The ideal genetic manipulation for treatment of a genetic disease would be the actual replacement of the defective gene with a normal copy of the gene. Homologous recombination is the term used for switching out a section of DNA and replacing it with a new piece. By this technique, the defective gene may be replaced with a normal gene which expresses a functioning BRCA2 tumor growth inhibitor protein.
• the growth of breast and ovarian cancer may be arrested or prevented by directly increasing the BRCA2 protein level where inadequate functional BRCA2 activity is responsible for breast and ovarian cancer.
• the cDNA and amino acid sequences of five novel BRCA2 haplotypes are disclosed herein (SEQ ID No:4-13). All or a fragment of BRCA2 protein may be used in therapeutic or prophylactic treatment of breast and ovarian cancer. Such a fragment may have a similar biological function as the native BRCA2 protein or may have a desired biological function as specified below.
• BRCA2 polypeptides or their functional equivalents including homologous and modified polypeptide sequences are also within the scope of the present invention.
• Changes in the native sequence may be advantageous in producing or using the BRCA2 derived polypeptides or functional equivalents suitable for therapeutic or prophylactic treatment of breast and ovarian cancer.
• these changes may be desirable for producing resistance against in vivo proteolytic cleavage, for facilitating transportation and delivery of therapeutic reagents, for localizing and compartmentalizing tumor suppressing agents, or for expression, isolating and purifying the target species.
• an active BRCA2 polypeptide or a functional equivalent as a tumor growth inhibitor there are a variety of methods to produce an active BRCA2 polypeptide or a functional equivalent as a tumor growth inhibitor.
• one or more amino acids may be substituted, deleted, or inserted using methods well known in the art (Maniatis et al., 1982). Considerations of polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphiphathic nature of the amino acids play an important role in designing homologous polypeptide changes suitable for the intended treatment. In particular, conservative amino acid substitution using amino acids that are related in side-chain structure and charge may be employed to preserve the chemical and biological property.
• a homologous polyeptide typically contains at least 70% homology to the native sequence.
• Unnatural forms of the polypeptide may also be incorporated so long as the modification retains substantial biological activity.
• These unnatural polypeptides typically include structural mimics and chemical medications, which have similar three- dimensional structures as the active regions of the native BRCA2 protein.
• these modifications may include terminal D-amino acids, cyclic peptides, unnatural amino acids side chains, pseudopeptide bonds, N- terminal acetylation, glycosylation, and biotinylation, etc.
• These unnatural forms of polypeptide may have a desired biological function, for example, they may be particularly robust in the presence of cellular or serum proteases and exopeptidase.
• An effective BRCA2 polypeptide or a functional equivalent may also be recognized by the reduction of the native BRCA2 protein.
• Regions of the BRCA2 protein may be systematically deleted to identify which regions are essential for tumor growth inhibitor activity. These smaller fragments of BRCA2 protein may then be subjected to structural and functional modification to derive therapeutically or prophylactically effective regiments. Finally, drugs, natural products or small molecules may be screened or synthesized to mimic the function of the BRCA2 protein.
• the active species retain the essential three- dimensional shape and chemical reactivity, and therefore retain the desired aspects of the biological activity of the native BRCA2 protein.
• the activity and function of BRCA2 may include transactivation, granin, DNA repair among others. Functions of BRCA2 protein are also reviewed in Bertwistle and Ashworth, Curr. Opin. Genet. Dev.
• BRCA2 polypeptide or a functional equivalent may be selected because such polypeptide or functional equivalent possesses similar biological activity as the native BRCA2 protein.
• All or fragments of the BRCA2 protein and polypeptide may be produced by host cells that are capable of directing the replication and the expression of foreign genes.
• Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic cells, which contain an expression vector comprising all or a fragment of the BRCA2 cDNA sequence (SEQ. ID No: 4, 6, 8, 10, or 12) operatively linked to one or more regulatory sequences to produce the intended BRCA2 protein or polypeptide.
• Prokaryotes may include gram negative or gram positive organisms, for example E. coli or Bacillus strains.
• Suitable eukaryotic host cells may include yeast, virus, and malian systems. For example, Sf9 insect cells and human cell lines, such as COS, MCF7, HeLa, 293T, HBL100, SW480, and HCT116 cells.
• An expression vector typically contains an origin of replication, a promoter, a phenotypic selection gene (antibiotic resistance or autotrophic requirement), and a DNA sequence coding for all or fragments of the BRCA2 protein.
• the expression vectors may also include other operatively linked regulatory DNA sequences known in the art, for example, stability leader sequences, secretory leader sequences, restriction enzyme cleavage sequences, 5 polyadenylation sequences, and termination sequences, among others.
• the essential and regulatory elements of the expression vector must be compatible with the intended host cell.
• Suitable expression vectors containing the desired coding and control regions may be constructed using standard recombinant DNA techniques known in the art, many of which are 0 described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).
• suitable origins of replication may include Col E1 , SV4O viral and M13 origins of replication.
• Suitable promoters may be constitutive or inducible, for example, tac promoter, lac Z promoter, SV40 5 promoter, MMTV promoter, and LXSN promoter. Examples of selectable markers include neomycin, ampicillin, and hygromycin resistance and the like.
• BRCA2 protein or o polypeptide is produced as a fusion protein to enhance the expression in selected host cells, to detect the expression in transfected cells, or to simplify the purification process.
• Suitable fusion partners for the BRCA2 protein or polypeptide are well known in the art and may include ⁇ -galactosidase, glutathione-S-transferase, and poly-histidine tag.
• Expression vectors may be introduced into host cells by various methods known in the art. The transformation procedure used depends upon the host to be transformed. Methods for introduction of vectors into host cells may include calcium phosphate precipitation, electrosporation, dextran- mediated transfection, liposome encapsulation, nucleus microinjection, and o viral or phage infection, among others.
• the host cell may be cultured under conditions permitting expression of large amounts of the BRCA2 protein or polypeptide.
• the expression product may be identified by many approaches well known in the art, for example, sequencing after PCR-based amplification, hybridization using probes complementary to the desired DNA sequence, the presence or absence of marker gene functions such as enzyme activity or antibiotic resistance, the level of mRNA production encoding the intended sequence, immunological detection of a gene product using monoclonal and polyclonal antibodies, such as Western blotting or ELISA.
• the BRCA2 protein or polypeptides produced in this manner may then be isolated following cell lysis and purified using various protein purification techniques known in the art, for example, ion exchange chromatography, gel filtration chromatography and immunoaffinity chromatography.
• BRCA2 protein or polypeptide are used, particularly to include the desired functional domains of BRCA2 protein.
• Expression of shorter fragments of DNA may be useful in generating BRCA2 derived immunogen for the production of anti-BRCA2 antibodies.
• not all expression vectors, DNA regulatory sequences or host cells will function equally well to express the BRCA2 protein or polypeptides of the present invention.
• one of ordinary skill in the art may make a selection among expression vectors, DNA regulatory sequences, host cells, and codon usage in order to optimize expression using known technology in the art without undue experimentation.
• fragments of the BRCA2 protein or polypeptides be obtained by overexpression in prokaryotic or eukaryotic host cells
• the BRCA2 polypeptides or their functional equivalents may also be obtained by in vitro translation or synthetic means by methods known to those of ordinary skill in the art.
• in vitro translation may employ an mRNA encoded by a DNA sequence coding for fragments of the BRCA2 protein or polypeptides.
• Chemical synthesis methodology such as solid phase synthesis may be used to synthesize a BRCA2 polypeptide structural mimic and chemically modified analogs thereof.
• the polypeptides or the modifications and mimic thereof produced in this manner may then be isolated and purified using various purification techniques, such as chromatographic procedures including ion exchange chromatography, gel filtration chromatography and immunoaffinity chromatography.
• BRCA2 protein targeted therapies may be utilized in treating and preventing tumors in breast and ovarian cancer.
• the present invention therefore includes therapeutic and prophylactic treatment of breast and ovarian cancer using therapeutic pharmaceutical compositions containing the BRCA2 protein, polypeptides, or their functional equivalents.
• protein replacement therapy may involve directly administering the BRCA2 protein, a BRCA2 polypeptide, or a functional equivalent in a pharmaceutically effective carrier.
• protein replacement therapy may utilize tumor antigen specific antibody fused to fragments of the BRCA2 protein, a polypeptide, or a functional equivalent to deliver anti-cancer regiments specifically to the tumor cells.
• an active BRCA2 protein, a BRCA2 polypeptide, or its functional equivalent is combined with a pharmaceutical carrier selected and prepared according to conventional pharmaceutical compounding techniques.
• a suitable amount of the composition may be administered locally to the site of a tumor or systemically to arrest the proliferation of tumor cells.
• the methods for administration may include parenteral, oral, or intravenous, among others according to established protocols in the art.
• compositions which may be added to enhance or stabilize the composition, or to facilitate preparation of the composition include, without limitation, syrup, water, isotonic solution, 5 % glucose in water or buffered sodium or ammonium acetate solution, oils, glycerin, alcohols, flavoring agents, preservatives, coloring agents, starches, sugars, diluents, granulating agents, lubricants, binders, and sustained release materials.
• the dosage at which the therapeutic compositions are administered may vary within a wide range and 5 depends on various factors, such as the stage of cancer progression, the age and condition of the patient, and may be individually adjusted.
• the BRCA2 protein, polypeptides, their functional equivalents, 0 antibodies, and polynucleotides may be used in a wide variety of ways in addition to gene therapy and protein replacement therapy. They may be useful as diagnostic reagents to measure normal or abnormal activity of BRCA2 at the DNA, RNA, and protein level.
• the present invention therefore encompasses the diagnostic reagents derived from the BRCA2 cDNA and 5 protein sequences as set forth in SEQ. ID. Nos: 4-13. These reagents may be utilized in methods for monitoring disease progression, for determining patients suited for gene and protein replacement therapy, or for detecting the presence or quantifying the amount of a tumor growth inhibitor following such therapy.
• Such methods may involve conventional histochemical o techniques, such as obtaining a tumor tissue from the patient, preparing an extract and testing this extract for tumor growth or metabolism.
• the test for tumor growth may involve measuring abnormal BRCA2 activity using conventional diagnostic assays, such as Southern, Northern, and Western blotting, PCR, RT-PCR, and immunoprecipitation.
• diagnostic assays such as Southern, Northern, and Western blotting, PCR, RT-PCR, and immunoprecipitation.
• the loss of BRCA2 expression in tumor tissue may be verified by RT-PCR and Northern blotting at the RNA level.
• a Southern blot analysis, genomic PCR, or fluorescence in situ hybridization (FISH) may also be performed to examine the mutations of BRCA2 at the DNA level.
• FISH fluorescence in situ hybridization
• a Western blotting, protein truncation assay, or immunoprecipitation may be o utilized to analysis the effect at the protein level.
• diagnostic reagents are typically either covalently or non convalently attached to a detectable label.
• a label includes a radioactive label, a colorimetric enzyme label, a fluorescence label, or an epitope label.
• a reporter gene downstream of the regulatory sequences is fused with the BRCA2 protein or polypeptide to facilitate the detection and purification of the target species.
• Commonly used reporter genes in BRCA2 fusion proteins include ⁇ -galactosidase and luciferase gene.
• the BRCA2 protein, polypeptides, their functional equivalents, antibodies, and polynucleotides may also be useful in the study of the characteristics of BRCA2 proteins, such as structure and function of BRCA2 in oncogenesis or subcellular localization of BRCA2 protein in normal and cancerous cell.
• yeast two-hybrid system has been used in the study of cellular function of BRCA2 to identify the regulator and effector of BRCA2 tumor suppressing function (Sharan et al., Nature 386:804-810 (1997) and Katagiri et al., Genes, Chromosomes & Cancer 21:217 ' -222 (1988)).
• BRCA2 protein, polypeptides, their functional equivalents, antibodies, and polynucleotides may also be used in in vivo cell based and in vitro cell free assays to screen natural products and synthetic compounds which may mimic, regulate or stimulate BRCA2 protein function.
• Antisense suppression of endogenous BRCA2 expression may assess the effect of BRCA2 protein on cell growth inhibition using known method in the art (Crooke, Annu. Rev. Pharmacol. Toxicol. 32:329-376 (1992) and Robinson-Benion and Holt, Methods Enzymol. 254:363-375 (1995)). Given the cDNA sequence as set forth in SEQ ID. NO: 4, 6, 8, 10, and 12, one of skill in the art can readily obtain anti-sense strand of DNA and RNA sequences to interfere with the production of wild-type BRCA2 protein or the mutated form of BRCA2 protein. Alternatively, antisense oligonucleotide may be designed to target the control sequences of BRCA2 gene to reduce or prevent the expression of the endogenous BRCA2 gene.
• the BRCA2 protein, polypeptides, or their functional equivalents may be used as immunogens to prepare polyclonal or monoclonal antibodies capable of binding the BRCA2 derived antigens in a known manner (Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988). These antibodies may be used for the detection of the BRCA2 protein, polypeptides, or a functional equivalent in an immunoassay, such as ELISA, Western blot, radioimmunoassay, enzyme immunoassay, and immunocytochemistry.
• an anti-BRCA2 antibody is in solution or is attached to a solid surface such as a plate, a particle, a bead, or a tube.
• the antibody is allowed to contact a biological sample or a blot suspected of containing the BRCA2 protein or polypeptide to form a primary immunocomplex. After sufficient incubation period, the primary immunocomplex is washed to remove any non-specifically bound species.
• the amount of specifically bound BRCA2 protein or polypeptide may be determined using the detection of an attached label or a marker, such as a radioactive, a fluorescent, or an enzymatic label.
• the detection of BRCA2 derived antigen is allowed by forming a secondary immunocomplex using a second antibody which is attached with a such label or marker.
• the antibodies may also be used in affinity chromatography for isolating or purifying the BRCA2 protein, polypeptides or their functional equivalents.
• a first degree relative is a parent, sibling, or offspring.
• a second degree relative is an aunt, uncle, grandparent, grandchild, niece, nephew, or half-sibling. Genomic DNA was isolated from white blood cells of five normal subjects selected from analysis of their answers to the questions above. Dideoxy sequence analysis was performed following polymerase chain reaction amplification.
• Taq Dye Terminator Kit Perkin-Elmer® cat# 401628. DNA sequencing was performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) automated sequencer (Model 377). The software used for analysis of the resulting data was "Sequence Navigator" purchased through ABI.
• Genomic DNA (100 nanograms) extracted from white blood cells of five normal subjects. Each of the five samples was sequenced end to end. Each sample was amplified in a final volume of 25 microliters containing 1 microliter (100 nanograms) genomic DNA, 2.5 microliters 10X PCR buffer (100 mM Tris, pH 8.3, 500 mM KCI, 1.2 mM MgCI 2 ), 2.5 microliters 10X dNTP mix (2 mM each nucleotide), 2.5 microliters forward primer, 2.5 microliters reverse primer, and 1 microliter Taq polymerase (5 units), and 13 microliters of water.
• 10X PCR buffer 100 mM Tris, pH 8.3, 500 mM KCI, 1.2 mM MgCI 2
• 2.5 microliters 10X dNTP mix (2 mM each nucleotide
• 2.5 microliters forward primer 2.5 microliters reverse primer
• 1 microliter Taq polymerase 5 units
• the primers in TABLE II below were used to carry out amplification of the various sections of the BRCA2 gene samples.
• the primers were synthesized on an DNA/RNA Synthesizer Model 394®.
• the BIC BRCA2 sequence also contains sequence errors in which a strech of nine nucleotides at positions 5554-5460 is listed as CGTTTGTGT (amino acids: Arg-Leu-Cys). The correct sequence at these positions is GTTTGTGTT (amino acids: Val-Cys-Val).
• the BIC BRCA2 nuclotides at positions 2024 are T, T, A, C, and T respectively, wherein the correct nucleotides at these positions are C, C, G, T, and C respectively.
• the nuclotide errors at codon 599, 1442, 1915 result in amino acids changes. Additional differences in the nucleic acids of the five normal individuals were found in ten polymorphic locations. The changes and their positions are found in TABLE III.
• the individual haplotypes of each chromosome of BRCA2 are displayed in FIGURE 3.
• the initial haplotype reported in Genbank was subtracted to determine the new haplotypes OMI 1-5.
• the Genbank sequence only represents 50% of the haplotypes found; the five new BRCA2 (om ⁇ 1 5) DNA sequences are shown as SEQ. ID. NO: 4, 6, 8, 10, and 12, respectively (See FIGURE 3), and the corresponding polypeptides are listed as SEQ. ID. NO: 5, 7, 9, 11 , and 13 respectively.
• these seven haplotypes represent a functional allele profile for the BRCA2 gene.
• Part A Answer the following questions about your family
• FAP Familial Adenomatous Polyposis
• Part B Refer to the list of cancers below for your responses only to questions in Part B
• Part C Refer to the list of relatives below for responses only to questions in Part C
• Part D Refer to the list of relatives below for responses only to questions in Part D.
• Polymorphisms For Reference A person skilled in the art of genetic susceptibility testing will find the present invention useful for: a) identifying individuals having a normal BRCA2 gene; b) avoiding misinterpretation of normal polymorphisms found in the normal population. Sequencing was carried out as in EXAMPLE 1 using a blood sample from the
• PCR primers used to amplify a patient's sample BRCA2 gene are listed in TABLE II.
• the primers were synthesized on a DNA/RNA Synthesizer Model
• 394® Thirty-five cycles are of amplification are performed, each consisting of denaturing (95°C; 30 seconds), annealing (55°C; 1 minute), and extension (72°C; 90 5 seconds), except during the first cycle in which the denaturing time is increased to 5 minutes and during the last cycle in which the extension time is increased to 5 minutes.
• PCR products are purified using Qia-quick® PCR purification kits (Qiagen®, cat# 28104; Chatsworth, CA). Yield and purity of the PCR product are determined o spectrophotometrically at OD260 on a Beckman DU 650 spectrophotometer.
• Fluorescent dye is attached to PCR products for automated sequencing using the Taq Dye Terminator Kit (Perkin-Elmer® cat# 401628). DNA sequencing is 5 performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) Foster City, CA., automated sequencer (Model 377).
• the software used for analysis of the resulting data is "Sequence Navigator®” purchased through ABI.
• the BRCA2 (o i 1 5) sequences were entered sequentially into the Sequence Navigator software as the standards for comparison.
• the Sequence Navigator software 0 compares the patient sample sequence to each BRCA2 (omi 1 5) standard, base by base. The Sequence Navigator highlights all differences between the standards (omi 1-5) and the patient's sample sequence.
• a first technologist checks the computerized results by comparing visually the BRCA2 (o i 1 - 5 ' standards against the patient's sample, and again highlights any differences between the standard and the sample.
• the first primary technologist 5 interprets the sequence variations at each position along the sequence. Chromatograms from each sequence variation are generated by the Sequence Navigator and printed on a color printer. The peaks are interpreted by the first primary technologist and a second primary technologist.
• a secondary technologist then reviews the chromatograms. The results are finally interpreted by a geneticist. 0 In each instance, a variation is compared to known normal polymorphisms for position and base change.
• the patient's BRCA2 sequence was found to be heterozygous at seven 5 nucleotide positions: 1093 (A/C), 1342 (A/C), 1593 (A/G), 2457 (C/T), 2908 (A/G), 3199 (A/G) and 9079 (A G).
• a person skilled in the art of genetic susceptibility testing will find the present invention useful for determining the presence of a known or previously unknown o mutation in the BRCA2 gene.
• a list of mutations of BRCA2 is publicly available in the Breast Cancer Information Core at http://www.nchgr.nih.gov/dir/lab_transfer/bic. This data site became publicly available on November 1 , 1995. Friend, S. et al. Nature Genetics 11:238, (1995).
• a mutation in exon 11 is characterized by amplifying the region of the mutation with a primer set which amplifies the region of the mutation. Sequencing was carried out as in Example 1 using a blood sample from the patient in question.
• exon 11 of the BRCA2 gene is subjected to direct dideoxy sequence analysis by asymmetric amplification using the polymerase chain reaction (PCR) to generate a single stranded product amplified from this DNA sample.
• PCR polymerase chain reaction
• Genomic DNA (100 nanograms) extracted from white blood cells of the subject is amplified in a final volume of 25 microliters containing 1 microliter (100 nanograms) genomic DNA, 2.5 microliters 10X PCR buffer (100 mM Tris, pH 8.3, 500 mM KCI, 1.2 mM MgCI 2 ), 2.5 microliters 10X dNTP mix (2 mM each nucleotide),
• PCR primers used to amplify segment Q of exon 11 are as follows:
• the primers are synthesized on an DNA/RNA Synthesizer Model 394®. Thirty-five cycles are performed, each consisting of denaturing (95°C; 30 seconds), annealing (55°C; 1 minute), and extension (72°C; 90 seconds), except during the first cycle in which the denaturing time is increased to 5 minutes, and during the last cycle in which the extension time is increased to 5 minutes.
• PCR products are purified using Qia-quick® PCR purification kits (Qiagen®, cat# 28104; Chatsworth, CA). Yield and purity of the PCR product are determined spectrophotometrically at OD260 on a Beckman DU 650 spectrophotometer.
• Fluorescent dye is attached to PCR products for automated sequencing using the Taq Dye Terminator Kit (Perkin-Elmer® cat# 401628). DNA sequencing is performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) Foster City, CA., automated sequencer (Model 377). The software used for analysis of the resulting data is "Sequence Navigator®" purchased through ABI.
• the BRCA2 (omi 1"5) sequence is entered into the Sequence Navigator software as the Standard for comparison.
• the Sequence Navigator software compares the sample sequence to the BRCA2 (o i) standard, base by base.
• the Sequence Navigator highlights all differences between the BRCA2 (omi) normal DNA sequence and the patient's sample sequence.
• a first technologist checks the computerized results by comparing visually the BRCA2 (omi 1 5) standard against the patient's sample, and again highlights any differences between the standard and the sample. The first primary technologist then interprets the sequence variations at each position along the sequence.
• the 6174delT mutation may be found. Mutations are noted by the length of non-matching sequence variation. Such a lengthy mismatch pattern occurs with deletions and insertions.
• This mutation is named in accordance with the suggested nomenclature for naming mutations, Beaudet, A et al., Human Mutation 2:245-248, (1993).
• the 6174delT mutation at codon 1982 of the BRCA2 gene lies in segment "Q" of exon 11.
• the DNA sequence results demonstrate the presence of a one base pair deletion of a T at nucleotide 6174 of the BRCA2 (omM 5) sequences.
• This mutation interrupts the normal reading frame of the BRCA2 transcript, resulting in the appearance of an in-frame terminator (TAG) at codon position 2003. This mutation is, therefore, predicted to result in a truncated, and most likely, nonfunctional protein.
• TAG in-frame terminator
• DNA primers are used to amplify a fragment of BRCA2 using PCR technology.
• the product is then digested with suitable restriction enzymes and fused in frame with the gene encoding glutathione S-transferase (GST) in Eschehchia coli using GST expression vector pGEX (Pharmacia Biotech Inc.)
• GST expression vector pGEX GST expression vector pGEX (Pharmacia Biotech Inc.)
• the expression of the fusion protein is induced by the addition of isopropyl- ⁇ - thiogalactopyranoside.
• the bacteria are then lysed and the overexpressed fusion protein is purified with glutathione-sepharose beads.
• the fusion protein is then verified by SDS/PAGE gel and N-terminus protein sequencing.
• the purified protein is used to immunize rabbits according to standard procedures described in Harlow & Lane (1988). Polycolonal antibody is collected from the serum several weeks after and purified using known methods in the art. Monoclonal antibodies against all or fragments of BRCA2 protein, polypeptides, or functional equivalents are obtained using hybridoma technology, see also Harlow & Lane (1988).
• the BRCA2 protein or polypeptide is coupled to the carrier keyhole limpet hemocyanin in the presence of glutaraldehyde.
• the conjugated immunogen is mixed with an adjuvant and injected into rabbits. Spleens from antibody-containing rabbits are removed.
• the B-cells isolated from spleen are fused to myeloma cells using polyethylene glycol (PEG) to promote fusion.
• PEG polyethylene glycol
• the hybrids between the myeloma and B-cells are selected and screened for the production of antibodies to immunogen BRCA2 protein or polypeptide. Positive cells are recloned to generate monoclonal antibodies.
• BRCA2 in human tissues is determined using Northern blot analysis.
• Human tissues include those from pancreas, testis, prostate, ovary, breast, small intestine, and colon are obtained from Clontech Laboratories, Inc., Palo Alto, CA.
• the poly(A)+ mRNA Northern blots from different human tissues is hybridized to BRCA2 cDNA probes according to manufacture protocol.
• the expression level is further conformed by RT-PCR using oligo-d(T) as a primer and other suitable primers.
• RNA is prepared by lysing cell in the presence of guanidinium isocyanate.
• Poly(A) + mRNA is isolated using the PolyATract mRNA isolation system from Promega, Madison, Wl. The isolated RNA is then electrophoresed under denaturing conditions and transferred to Nylon membrane.
• the probe used for Northern blot is a fragment of BRCA2 sequence obtained by PCR amplification. The probes are labeled with [ ⁇ - 32 P] dCTP using a random-primed labeling kit (Amersham Life Science, Arlington Heights, IL).
• the whole-cell extracts of BRCA2 transfected cells are subjected to immunoprecipitation and immunoblotting to determine the BRCA2 protein level.
• the BRCA2 protein or polypeptide is immunoprecipitated using anti-BRCA2 antibodies prepared according to Example 4. Samples are then fractionated using SDS/PAGE gel and transferred to nitrocellulose. Western blot of the BRCA2 protein or polypeptide is performed with the indicated antibodies. Antibody reaction is revealed using enhanced chemiluminescence reagents (Dupont New England Nuclear, Boston, MA).
• the growth of ovarian or breast cancer may be arrested by increasing the expression of the BRCA2 gene where inadequate expression of that gene is responsible for hereditary ovarian or breast cancer.
• Gene therapy may be performed on a patient to reduce the size of a tumor.
• the LXSN vector may be transformed with a BRCA2 (om ⁇ 1 5) coding sequence as presented SEQ ID NO:4, 6, 8, 10, or 12 or a fragment thereof.
• the LXSN vector is transformed with a fragment of the wildtype BRCA2 (om ⁇ 1 - 5) coding sequence as set forth in SEQ ID NO:4, 6, 8, 10, or 12.
• the LXSN-BRCA2 (om ⁇ 1 - 5) retroviral expression vector is constructed by cloning a Sal I linkered BRCA2 (om ⁇ 1 5) cDNA or fragments thereof into the Xho I site of the vector LXSN. Constructs are confirmed by DNA sequencing. See Holt et al., Nature Genetics 12: 298-302 (1996).
• Retroviral vectors are manufactured from viral producer cells using serum free and phenol-red free conditions and tested for sterility, absence of specific pathogens, and absence of replication-competent retrovirus by standard assays. Retrovirus is stored frozen in aliquots which have been tested.
• Patients receive a complete physical exam, blood, and urine tests to determine overall health. They may also have a chest X-ray, electrocardiogram, and appropriate radiologic procedures to assess tumor stage. Patients with metastatic ovarian cancer are treated with retroviral gene therapy by infusion of recombinant LXSN-BRCA2 (om ⁇ 1 5) retroviral vectors into peritoneal sites containing tumor, between 10 9 and 10 10 viral particles per dose. 5 Blood samples are drawn each day and tested for the presence of retroviral vector by sensitive polymerase chain reaction (PCR)-based assays. The fluid which is removed is analyzed to determine:
• PCR polymerase chain reaction
• RT-PCR is performed with by the method of Thompson et al., Nature Genetics 9: 444-450 (1995), using primers derived from a BRCA2 (om ⁇ 1 5) coding sequence as in SEQ ID NO:4, 6, 8, 10, or 12 or fragments thereof.
• Cell lysates are prepared and immunoblotting is performed by the method of Jensen et al., Nature 5 Genetics 12: 303-308 (1996) and Jensen et al., Biochemistry 3 . : 10887-10892 (1992).
• LXSN-BRCA2 (om ⁇ 1 5) patients with measurable disease are also evaluated for a clinical response to LXSN-BRCA2 (om ⁇ 1 5) especially those that do not undergo a palliative intervention immediately after retroviral vector therapy. Fluid cytology, abdominal girth, CT scans of the abdomen, and local symptoms are followed. 5
• Partial Response decrease of at least 50% of the sum of the products of 0 the 2 largest perpendicular diameters of all measurable lesions as determined by 2 observations not less than 4 weeks apart. To be considered a PR, no new lesions should have appeared during this period and none should have increased in size.
• Stable Disease less than 25% change in tumor volume from previous evaluations.
• Progressive Disease greater than 25% increase in tumor measurements from prior evaluations. The number of doses depends upon the response to treatment.
• Therapeutically elevated level of functional BRCA2 protein may alleviate the absence or reduced endogenous BRCA2 tumor suppressing activity.
• Breast or ovarian cancer is treated by the administration of a therapeutically effective amount 0 of the BRCA2 protein, a polypeptide, or its functional equivalent in a pharmaceutically acceptable carrier.
• Clinically effective delivery method is applied either locally at the site of the tumor or systemically to reach other metastasized locations with known protocols in the art. These protocols may employ the methods of direct injection into a tumor or diffusion using time release capsule.
• a 5 therapeutically effective dosage is determined by one of skill in the art.
• Breast or ovarian cancer may be prevented by the administration of a prophylactically effective amount of the BRCA2 protein, polypeptide, or its functional equivalent in a pharmaceutically acceptable carrier.
• Individuals with known risk for breast or ovarian cancer are subjected to protein replacement therapy to prevent o tumorigenesis or to decrease the risk of cancer.
• Elevated risk for breast and ovarian cancer includes factors such as carriers of one or more known BRCA1 and BRCA2 mutations, late child bearing, early onset of menstrual period, late occurrence of menopause, and certain high risk dietary habits.
• Clinically effective delivery method is used with known protocols in the art, such as administration into peritoneal cavity, 5 or using an implantable time release capsule.
• a prophylactically effective dosage is determined by one of skill in the art.
• TELECOMMUNICATION INFORMATION (A) TELEPHONE: 650-463-8109 (B) TELEFAX: 650-463-8400
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE Genomic DNA
• FEATURE (A) NAME/KEY: exon
• ACAGATTTGT G ACCGGCGCG GTTTTTGTCA GCTTACTCCG GCCAAAAAAG AACTGCACCT 180 CTGGAGCGGA CTTATTTACC AAGCATTGGA GGAATATCGT AGGTAAAA ATG CCT ATT 237
• TGT CCA CTT CTA AAT TCT TGT CTT AGT GAA AGT CCT GTT GTT CTA CAA 669
• Glu Ser Asp Val Glu Leu Thr Lys Asn lie Pro Met Glu Lys Asn Gin 805 810 815 GAT GTA TGT GCT TTA AAT GAA AAT TAT AAA AAC GTT GAG CTG TTG CCA 2733
• AAC ACT CAG 4365 lie Cys Leu Lys Leu Ser Gly Gin Phe Met Lys Glu Gly Asn Thr Gin 1365 1370 1375 ATT AAA GAA GAT TTG TCA GAT TTA ACT TTT TTG GAA GTT GCG AAA GCT 4413 lie Lys Glu Asp Leu Ser Asp Leu Thr Phe Leu Glu Val Ala Lys Ala 1380 1385 1390 1395
• Gly Gin Pro Glu Arg lie Asn Thr Ala Asp Tyr Val Gly Asn Tyr Leu 1700 1705 1710 1715 TAT GAA AAT AAT TCA AAC AGT ACT ATA GCT GAA AAT GAC AAA AAT CAT 5421
• GAG GAA ATG GTT TTG TCA AAT TCA AGA ATT GGA AAA
• AGA AGA GGA GAG 7053 Glu Glu Met Val Leu Ser Asn Ser Arg lie Gly Lys Arg Arg Gly Glu
• GCA AAA TAT GTG GAG GCC CAA CAA AAG AGA CTA GAA GCC TTA TTC ACT 8829 Ala Lys Tyr Val Glu Ala Gin Gin Lys Arg Leu Glu Ala Leu Phe Thr 2855 2860 2865
• Lys Glu Lys Asp Ser Val lie Leu Ser lie Trp Arg Pro Ser Ser Asp 2980 2985 2990 2995 TTA TAT TCT CTG TTA ACA GAA GGA AAG AGA TAC AGA ATT TAT CAT CTT 9261 Leu Tyr Ser Leu Leu Thr Glu Gly Lys Arg Tyr Arg lie Tyr His Leu 3000 3005 3010
• AAA AGG AAG TCT GTT TCC ACA CCT GTC TCA GCC CAG ATG ACT TCA AAG 9981 Lys Arg Lys Ser Val Ser Thr Pro Val Ser Ala Gin Met Thr Ser Lys
• Lys Lys He Met Glu Arg Asp Asp Thr Ala Ala Lys Thr Leu Val Leu 2675 2680 2685 Cys Val Ser Asp He He Ser Leu Ser Ala Asn He Ser Glu Thr Ser 2690 2695 2700
• TGT CCA CTT CTA AAT TCT TGT CTT AGT GAA AGT CCT GTT GTT CTA CAA 669 Cys Pro Leu Leu Asn Ser Cys Leu Ser Glu Ser Pro Val Val Leu Gin 135 140 145
• GGT ATC AAA AAG TCT ATA TTC AGA ATA AGA GAA TCA CCT AAA GAG ACT 1773 Gly He Lys Lys Ser He Phe Arg He Arg Glu Ser Pro Lys Glu Thr 500 505 510 515
• AAA AGA AGC TGT TCA CAG AAT GAT TCT GAA GAA CCA ACT TTG TCC TTA 2205 Lys Arg Ser Cys Ser Gin Asn Asp Ser Glu Glu Pro Thr Leu Ser Leu 645 650 655
• GCC TGT AAA GAC CTT GAA TTA GCA TGT GAG ACC ATT GAG ATC ACA GCT 4989 Ala Cys Lys Asp Leu Glu Leu Ala Cys Glu Thr He Glu He Thr Ala 1575 1580 1585 GCC CCA AAG TGT AAA GAA ATG CAG AAT TCT CTC AAT AAT GAT AAA AAC 5037

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