EP1165779A1 - Ein presenilin assoziiertes membran protein und verwendungen davon - Google Patents
Ein presenilin assoziiertes membran protein und verwendungen davonInfo
- Publication number
- EP1165779A1 EP1165779A1 EP00915062A EP00915062A EP1165779A1 EP 1165779 A1 EP1165779 A1 EP 1165779A1 EP 00915062 A EP00915062 A EP 00915062A EP 00915062 A EP00915062 A EP 00915062A EP 1165779 A1 EP1165779 A1 EP 1165779A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- pamp
- protein
- presenilin
- nucleic acid
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Definitions
- the present invention relates generally to the field of neurological and physiological dysfunctions associated with neuropsychiatric and developmental diseases, especially Alzheimer's Disease. More particularly, the invention is concerned with the identification of proteins associated with neuropsychiatric and developmental diseases, especially Alzheimer's Disease, and relates to methods of diagnosing these diseases, and to methods of screening for candidate compounds which modulate the interaction of a certain protein, specifically Presenilin Associated Membrane Protein ("PAMP”), with presenilin proteins.
- PAMP Presenilin Associated Membrane Protein
- AD Alzheimer's Disease
- the disease is accompanied by a constellation of neuropathologic features principal amongst which are the presence of extracellular amyloid or senile plaques, and neurofibrillary tangles in neurons.
- the etiology of this disease is complex, although in some families it appears to be inherited as an autosomal dominant trait.
- ⁇ -amyloid precursor protein ⁇ APP
- ⁇ APP ⁇ -amyloid precursor protein
- Abnormal processing of ⁇ APP with overproduction of amyloid- ⁇ is also a feature of other CNS diseases, including inherited and sporadic forms of amyloid angiopathy, which presents with i ⁇ tracerebral bleeding (Levy et al., Science 1990;248:1124-1126).
- abnormalities of presenilin proteins and PS-interacting proteins may affect these diseases as well.
- the presenilin genes encode homologous polytopic transmembrane proteins that are expressed at low levels in intracellular membranes including the nuclear envelope, the endoplasmic reticulum, the Golgi apparatus and some as yet uncharacte ⁇ zed intracytoplasmic vesicles in many different cell types including neuronal and non-neuronal cells (Sher ⁇ ngton et al , supra, Rogaev et al , supra, Levy-Lahad et al , supra, Doan et al , Neuron 1996,17 1023-1030, Walter et al , Molec Medicine 1996,2 673-691 , De Strooper et al , J Biol Chem 1997,272 3590-3598, Lehmann ef a/ , J Biol Chem 1997,272 12047-12051 , Li et al , Cell 1997,90 917-927) Structural studies predict that the following neuronal and non-neuronal cells (S
- Missense mutations in the PS1 and PS2 genes are associated with the inherited forms of early-onset AD (Shemngton et al , Nature 1995,375 754-760, Rogaev, et al , Nature 1995,376 775-778, Levy-Lahad et al, Science 1995,269 970-973)
- ⁇ APP ⁇ -amyloid precursor protein
- PS1 and PS2 interact specifically with at least two members of the armadillo family of proteins, neuronal plakophi n-related armadillo protein (Paffenholtz et al , Differentiation 1997, 61 293-304, Paffenholtz et al , Exp Cell Res 1999, 250 452-464, Zhou et al , Neuroreport 1997, 8 2085-2090) and ⁇ -catenin, that are expressed in both embryonic and post-natal tissues Moreover, the domains of PS1 and PS2 that interact with these proteins have been identified Mutations in PS1 and PS2 affect the translocation of ⁇ - catenin into the nucleus of both native cells and cells transfected with a mutant PS gene These interactions and effects are described in detail in co-pending commonly assigned U S Application Serial No 09/227,725, filed January 8, 1999, the disclosure of which is incorporated herein by reference The identification and cloning of normal as well as mutant PS1 and PS2 genes and gene products are described in detail in co
- AD Alzheimer's disease
- PS1 and PS2 interact specifically with a transmembrane protein, herein referred to as "Presenilin Associated Membrane Protein” or "PAMP", which is expressed in multiple tissues (e g , brain, kidney, lung, etc )
- PAMP Presenilin Associated Membrane Protein
- the product of this gene is therefore implicated in the biochemical pathways affected in Alzheimer's Disease, and may also have a role in other dementias, amyloid angiopathies, and developmental disorders such as spma bifida
- This gene therefore, presents a new therapeutic target for the treatment of Alzheimer's Disease and other neurologic diseases
- PAMP nucleic acids, proteins and peptides, antibodies to PAMP, cells transformed with PAMP nucleic acids, and transgenic animals altered with PAMP nucleic acids that possess various utilities, as described herein for the diagnosis, therapy and continued investigation of Alzheimer's Disease and other neurodegenerative disorders
- the invention provides isolated and purified presenilin associated membrane protein (PAMP), or a functional fragment thereof, as well as nucleic acids encoding a PAMP Preferred nucleotide and ammo acid sequences are provided herein
- PAMP presenilin associated membrane protein
- the invention further provides probes and primers for PAMP genes Preferred embodiments include sequences of at least 10, 15 or 20 consecutive nucleotides selected from the disclosed sequences
- the invention also provides isolated and purified mutant PAMP, or a functional fragment thereof, as well as nucleic acids encoding a mutant PAMP, and probes and primers for PAMP genes Preferred nucleotide and ammo acid sequences are provided herein
- allelic variants or heterospecfic homologues of a human PAMP and gene are provided The methods may be practiced using nucleic acid hybridization or amplification techniques, immunochemical techniques, or any other technique known in the art
- the allelic variants may include other normal human alleles as well as mutant alleles of PAMP genes which may be causative of Alzheimer's Disease or other CNS diseases
- the heterospecific homologues may be from other mammalian species, such as mice, rats, dogs, cats or non-human primates, or may be from invertebrate species, such as Drosophila melanogaster or Caenorhabditis elegans
- the invention also provides vectors, and particularly expression vectors (e g , cos-Tet vector), which include any of the above-de
- the invention further provides host cells and transgenic animals transformed with any of the above-described nucleic acids of the invention
- the host cells may be prokaryotic or eukaryotic cells and, in particular, may be gametes, zygotes, fetal cells, or stem cells useful in producing transgenic animal models
- the transgenic animal contains a transgene encoding a normal or mutant PAMP, which is expressed in neural cells such that expression can be detected, e g , by detecting PAMP, mRNA, or protein, and more preferably by detecting a neuroprotective or a neurodegenerative phenotype
- the animal might manifest one or more symptoms of a neurodegenerative disease
- the animal may be a vertebrate or an invertebrate
- the transgenic animal is a mouse, and the transgene encodes a human PAMP
- the transgenic animal may further comprise a second transgene encoding a normal or mutant PS1 , PS2, or ⁇ APP
- the invention provides an animal containing a nucleic acid that expresses a PAMP or a mutant PAMP at a higher or lower level relative to the expression level in a wild-type animal
- the animal may be prepared by homologous recombination-mediated targeting of endogenous PAMP nucleic acid
- the animal is prepared by translocation of P-elements or chemical mutagenesis
- the invention also provides a reconstituted system for measuring PAMP activity, comprising PAMP, a mutant PAMP, or functional fragments thereof, and a PAMP substrate
- the reconstituted system may be a whole cell
- the whole cell contains a first nucleic acid that expresses said PAMP and a second nucleic acid that expresses the substrate
- the substrate comprises PS1 protein, PS2 protein, ⁇ APP, or a surrogate synthetic substrate protein such as Notch, which undergoes proteolytic processing events similar to those of ⁇ APP (Haass C and Selkoe DJ Nature 1998, 391 387-390, De Strooper B, et al , Nature 1999, 398 518-522, Song W, et al , Proc Natl Acad Sci USA 1999, 96 6959-6963 .Struhl G and Greenwald I, Nature 1999, 398 522-525,Ye Y, et al , Nature 1999, 398 525-529 )
- the invention
- PAMP associated with Alzheimer's or a related neurological disorder comprising obtaining a nucleic acid sample from an individual diagnosed with or suspected of having a neurodegenerative disorder, and sequencing a gene encoding PAMP from said sample
- the invention also provides a method for diagnosing individuals predisposed to or having a neurodegenerative disorder, comprising obtaining a nucleic acid sample from an individual diagnosed with or suspected of having a neurodegenerative disorder, and sequencing a gene encoding PAMP from said sample
- the invention also provides a method for diagnosing individuals predisposed to or having a neurodegenerative disorder, comprising obtaining cells that contain nucleic acid encoding PAMP, and under non-pathological conditions, transcribe the nucleic acid, and measuring a level of transc ⁇ ptional activity of the nucleic acid
- the invention further provides a method for diagnosing individuals predisposed to or having a neurodegenerative disorder, comprising obtaining cells from an individual that express nucleic acid encoding PAMP, or isolating PAMP from said individual, and measuring
- the invention also provides a method for identifying putative agents having anti-neurodegenerative activity, comprising administering one or more putative agents to a transgenic animal and detecting a change in PAMP activity
- the invention also provides a method for identifying putative agents having anti-neurodegenerative activity, comprising adding one or more said agents to the reconstituted system described above, and detecting a change in PAMP activity.
- the invention also provides a method for identifying putative agents having anti-neurodegenerative activity, comprising adding one or more said agents to the complex described above, and detecting a conformational change in PAMP.
- the invention also provides a method for identifying proteins that interact with PAMP, comprising contacting said substance with the reconstituted system discussed above, and detecting a change in PAMP activity.
- the invention provides for a method for identifying substances that modulate PAMP activity, comprising contacting a sample containing one or more substances with PAMP, or a PAMP mutant, or functional fragments thereof, and a PAMP substrate, measuring PAMP activity, and determining whether a change in PAMP activity occurs.
- the substance is a PAMP inhibitor.
- the substance stimulates PAMP activity.
- FIGURE 1A and 1 B Predicted amino acid sequences for human (SEQ ID NO:14), mouse (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:14), mouse (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16), D.melanogaster (SEQ ID NO:16),
- Alzheimer's Disease further understanding of the development and progression of this disease, as well as other neurodegenerative diseases, requires a more complete understanding of the functions of the presenilins and other proteins with which they interact
- the present invention advantageously identifies such a protein
- PAMP The invention is based, in part, on the discovery of a novel Type
- PAMP Presenilin Associated Membrane Protein
- PAMP ProteinBank, Accession No Q92542, SEQ ID NO 14
- the nucleotide sequence (SEQ ID NO 13) of human PAMP predicts that the gene encodes a Type 1 transmembrane protein of 709 ammo acids (SEQ ID NO 14), the protein having a short hydrophilic C-termmus (-20 residues), a hydrophobic transmembrane domain (15-20 residues), and a longer N-termmal hydrophilic domain which contains several potentially functional sequence motifs as listed below in Table 1
- the PAMP sequence also contains a Trp-Asp (WD) repeat (residue 226), at least one "DTG” motif (residues 91 - 93) present in eukaryotic aspartyl proteases, as well as several "DTA/DTAE” motifs (residues 480 - 482, 504 - 506) present in viral aspartyl proteases
- Trp-Asp WD
- the invention is further based on the identification of conserved functional domains, based on comparison and evaluation of the predicted amino acid sequences of human (SEQ ID NO: 14), murine (SEQ ID NO: 16), D. melanogaster (SEQ ID NO: 18), and C. elegans (SEQ ID NO: 12) orthologues of PAMP.
- PAMP can be characterized by the presence of conserved structural features, relative to orthologues from D melanogaster 0 and C. elegans Nucleotide sequences encoding homologous hypothetical proteins exist in mice multiple EST, and C. elegans (GenBank, www.ncbi.nlm.nih.gov; Accession No.
- One domain has chemical similarities to cyclic nucleotide binding domains of other proteins, and may have some regulatory role on a potential complex formed between PS1 PAMP and the C-terminal fragment of ⁇ APP, derived either from ⁇ - or ⁇ -secretase. These putative functional domains are sites for therapeutic target development by deploying drugs which might interact with these sites to modulate ⁇ APP processing via this complex.
- PAMP also refers to functionally active fragments of the protein.
- Such fragments include, but are not limited to, peptides that contain an epitope, e.g., as determined by conventional algorithms such as hydrophilicity/hydrophobicity analysis for antibody epitopes, and amphipathicity or consensus algorithms for T cell epitopes (Spouge, et al., J. Immunol, 138:2204, 1987; Margalit, et ai, J. Immunol., 138:2213, 1987; Rothbard, Ann. Inst. Pateur., 137E:518, 1986; Rothbard and Taylor, EMBO J.; 7:93, 1988).
- a functionally active fragment of PAMP is a conserved domain, relative to the D. melanogaster and C. e/egans orthologues.
- a specific functionally active fragment of PAMP is a fragment that interacts with PS1 or PS2, or both.
- PAMP also encompasses naturally occurring variants, including other mammalian PAMPs (readily identified, as shown herein for murine PAMP, based on the presence of the structural features set forth above), allelic variants of PAMP from other human sources (including variants containing polymorphisms that are predictive of disease propensity or of response to pharmacological agents), and mutant forms of PAMP or PAMP genes that are associated with neurological diseases and disorders (such as spina bifida), particularly neurodegenerative diseases (such as AD). Also included are artificial PAMP mutants created by standard techniques such as site directed mutagenesis or chemical synthesis.
- a PAMP "substrate” may be a polypeptide or protein, or any other type of compound, with which PAMP interacts physiologically.
- PAMP substrates include PS1 , PS2, and ⁇ APP.
- a PAMP "ligand” may be a polypeptide, protein, lipid, carbohydrate, vitamin, mineral, amino acid, or any other type of compound which binds to PAMP. Hypothetically, PAMP may function as a receptor which modulates PS1/PS2/ ⁇ APP processing in response to signal (ligand) dependent interactions with PAMP.
- PAMP a nucleic acid molecule
- normal text e.g., PAMP indicates the polypeptide or protein.
- the term “about” or “approximately” means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
- the term about means within an order of magnitude of a given value, preferably within a multiple of about 5-fold, and more preferably within a factor of about 2-fold of a given value.
- an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
- an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.
- the isolated nucleic acid lacks one or more introns.
- Isolated nucleic acid molecules can be inserted into plasmids, cosmids, artificial chromosomes, and the like.
- a recombinant nucleic acid is an isolated nucleic acid.
- An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
- An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
- An isolated material may be, but need not be, purified.
- purified refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants.
- a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell;
- a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
- substantially free is used operationally, in the context of analytical testing of the material.
- purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
- host cell means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or functional assays, as described infra.
- a host cell has been "transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
- a cell has been "transformed” by exongenous or heterologous DNA when the transfected DNA is expressed and effects a function or phenotype on the cell in which it is expressed.
- expression system means a host cell transformed by a compatible expression vector and cultured under suitable conditions e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
- Proteins and polypeptides can be made in the host cell by expression of recombinant DNA.
- polypeptide refers to an amino acid-based polymer, which can be encoded by a nucleic acid or prepared synthetically. Polypeptides can be proteins, protein fragments, chimeric proteins, etc.
- protein refers to a polypeptide expressed endogenously in a cell, e g , the naturally occurring form (or forms) of the ammo acid-based polymer
- a "coding sequence” or a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, t e , the nucleotide sequence encodes an ammo acid sequence for that polypeptide, protein or enzyme
- a coding sequence for a protein may include a start codon (usually ATG) and a stop codon
- the coding sequences herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ⁇ bosome entry sites (IRES) and other ⁇ bosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like
- the nucleic acids may also be modified by many means known in the art Non-limiting examples of such modifications include
- a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence
- the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background
- a coding sequence is "under the control” or “operatively associated with” of transc ⁇ ptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which then may be trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence
- express and expression mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence
- a DNA sequence is expressed in or by a cell to form an "expression product" such as a protein
- the expression product itself e g the resulting protein, may also be said to be “expressed” by the cell
- transfection means the introduction of a foreign nucleic acid into a cell
- transformation means the introduction of a "foreign” (/ e extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence
- the introduced gene or sequence may also be called a "cloned", “foreign”, or “heterologous” gene or sequence, and may include regulatory or control sequences used by a cell's genetic machinery
- vector means the vehicle by which a DNA or RNA sequence (e g , a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e g , transcription and translation) of the introduced sequence
- Vectors include plasmids, phages, viruses, etc
- a "cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct " Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic
- a "knockout mammal” is a mammal (e.g., mouse) that contains within its genome a specific gene that has been inactivated by the method of gene targeting (see, e.g., US Patents No. 5,777,195 and No. 5,616,491 ).
- a knockout mammal includes both a heterozygote knockout (i.e., one defective allele and one wild-type allele) and a homozygous mutant.
- Preparation of a knockout mammal requires first introducing a nucleic acid construct that will be used to suppress expression of a particular gene into an undifferentiated cell type termed an embryonic stem cell. This cell is then injected into a mammalian embryo.
- a mammalian embryo with an integrated cell is then implanted into a foster mother for the duration of gestation.
- Zhou, et al. (Genes and Development, 9:2623-34, 1995) describes PPCA knock-out mice. Knockout mice can be used to study defects in neurological development or neurodegenerative diseases. Disease phenotypes that develop can provide a platform for further drug discovery.
- knockout refers to partial or complete suppression of the expression of at least a portion of a protein encoded by an endogenous DNA sequence in a cell.
- knockout construct refers to a nucleic acid sequence that is designed to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell.
- the nucleic acid sequence used as the knockout construct is typically comprised of (1 ) DNA from some portion of the gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell.
- the knockout construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to prevent or interrupt transcription of the native DNA sequence. Such insertion usually occurs by homologous recombination (i.e., regions of the knockout construct that are homologous to endogenous DNA sequences hybridize to each other when the knockout construct is inserted into the cell and recombine so that the knockout construct is incorporated into the corresponding position of the endogenous DNA).
- the knockout construct nucleic acid sequence may comprise 1 ) a full or partial sequence of one or more exons and/or introns of the gene to be suppressed, 2) a full or partial promoter sequence of the gene to be suppressed, or 3) combinations thereof.
- the knockout construct is inserted into an embryonic stem cell (ES cell) and is integrated into the ES cell genomic DNA, usually by the process of homologous recombination This ES cell is then injected into, and integrates with, the developing embryo Generally, for homologous recombination, the DNA will be at least about 1 kilobase (kb) in length and preferably 3-4 kb in length, thereby providing sufficient complementary sequence for recombination when the knockout construct is introduced into the genomic DNA of the ES cell
- kb kilobase
- a "knock-in" mammal is a mammal in which an endogenous gene is substituted with a heterologous gene or a modified variant of the endogenous gene (Roemer et al , New Biol 3 331 , 1991 )
- the heterologous gene is "knocked-in” to a locus of interest, for example into a gene that is the subject of evaluation of expression or function, thereby linking the heterologous gene expression to transcription from the appropriate promoter (in which case the gene may be a reporter gene, see Elefanty ef al , Proc Natl Acad Sci USA 95 11897,1998)
- This can be achieved by homologous recombination, transposon (Westphal and Leder, Curr Biol 7 530, 1997), using mutant recombination sites (Araki ef al , Nucleic Acids Res 25 868, 1997) or PCR (Zhang and Henderson, Biotechniques 25 784, 1998)
- heterologous refers to a combination of elements not naturally occurring
- heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell
- the heterologous DNA includes a gene foreign to the cell
- a heterologous expression regulatory element is a such an element operatively associated with a different gene than the one it is operatively associated with in nature
- an gene is heterologous to the recombinant vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed, e g , a CHO cell
- mutant and mutant mean any detectable change in genetic material, e g DNA, or any process, mechanism, or result of such a change This includes gene mutations, in which the structure (e g , DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e g , protein) expressed by a modified gene or DNA sequence
- variant may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc , i e , any kind of mutant "Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the ammo acid encoded at that position
- “Function-conservative variants” are those in which a given am o acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an ammo acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like)
- Ammo acids with similar properties are well known in the art
- arginme, histidine and lysine are hydrophilic-basic ammo acids and may be interchangeable
- isoleucine, a hydrophobic ammo acid may be replaced with leucine, methionine or valme
- Ammo acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or ammo acid sequence similarity between any two proteins of similar function may vary and may be, for example, from
- homologous in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e g , the immunoglobulin superfamily) and homologous proteins from different species (e g , myosm light chain, etc ) (Reeck et al , Cell 50 667, 1987) Such proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs Motif analysis can be performed using, for example, the program BLOCKS (www blocks fhcrc org)
- sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or ammo acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al , supra)
- sequence similarity when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin
- two DNA sequences are "substantially homologous" or “substantially similar” when at least about 80%, and most preferably at least about 90 or 95% of the nucleotides match over the defined length of the DNA sequences, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA St ⁇ der, etc Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system
- two am o acid sequences are "substantially homologous” or “substantially similar” when greater than 80% of the am o acids are identical, or greater than about 90% are similar (functionally identical)
- the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, ProteinPredict
- a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra).
- the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
- low stringency hybridization conditions corresponding to a T m (melting temperature) of 55 ⁇ C, can be used.
- Moderate stringency hybridization conditions correspond to a higher T m and high stringency hybridization conditions correspond to the highest T m .
- Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
- the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
- the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA.
- a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
- antisense nucleic acids including ribozymes
- An "antisense nucleic acid” is a single stranded nucleic acid molecule which, on hybridizing under cytoplasmic conditions with complementary bases in an RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid. As presently used, "antisense” broadly includes RNA-RNA interactions, RNA- DNA interactions, ribozymes and RNase-H mediated arrest.
- Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent No. 5,814,500; U.S. Patent No. 5,811 ,234), or alternatively they can be prepared synthetically (e.g., U.S. Patent No; 5,780,607).
- oligonucleotide refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.
- a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.
- oligonucleotides (one or both of which may be labeled) can be used as PCR primers, e.g., for cloning full length or a fragment of a protein or polypeptide.
- an oligonucleotide of the invention can form a triple helix with a nucleic acid (genomic DNA or mRNA) encoding a protein or polypeptide.
- oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer.
- oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
- the oligonucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly.
- Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
- synthetic oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphot esters, methyl phosphonates, short chain alkyl, or cycloalkl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
- PS1 and PS2 genes are described in Applicants' co-pending U S Application Serial No 08/888,077, filed July 3, 1997, and incorporated herein by reference
- PS1 mutations include I143T, M146L, L171 P, F177S, A260V, C263R, P264L, P267S, E280A, E280G, A285V, L286V, ⁇ 291-319, L322V, G384A, L392V, C410Y and I439V
- PS2 mutations include N141 I, M239V and I420T
- the methods of the present invention are not limited to mutant presenilins wherein the PAMP-mteracting domain is mutated relative to the wild-type protein
- Applicants have observed that mutations in PS1 (e g , M146L) outside of the interacting domain (loop) also affect ⁇ - catenm translocation These mutations probably disturb the presenilin armadill
- Proteins that interact with the presenilins, / e , PS-interacting proteins include PAMP, the S5a subunit of the 26S proteasome (GenBank, Accession No U51007), Rab11 (GenBank, Accession Nos X56740 and X53143), retmoid X receptor B, also known as nuclear receptor co-regulator or MHC (GenBank Accession Nos M84820, and X63522), GT24 (GenBank Accession No U81004), ⁇ -catenm (Zhou et al , 1997, Neuro Report (Fast Track) 8 1025-1029 and Yu et al , supra) as well as armadillo proteins
- PS1 binding proteins are described in Applicants' copending commonly assigned U S Application Serial No 08/888,077, filed July 3, 1997, as well as U S Application Serial No 08/592,541 , filed January 26, 1996, the disclosures of which are incorporated herein by reference
- PAMP Mutants PAMP mutants may cause biochemical changes similar to those affecting the onset or progression of Alzheimer Disease Therefore, artificial PAMP mutations can potentially be used to generate cellular and other model systems to design treatments and preventions for Alzheimer Disease related disorders Such mutations may also be used for evaluating whether PAMP is involved in the pathogenesis of AD Since the amyloid- ⁇ (A ⁇ ) inducing mutations are found in ammo acid residues of a soluble (non-membrane spanning) domain of PAMP, analysis of the normal structure of this domain and the effects of these and other nearby mutations on the structure of this domain (and the other domains of PAMP) provide information for the design of specific molecular therapeutics In general, modifications of the sequences encoding the polypeptides described herein may be readily accomplished by standard techniques such as chemical syntheses and site-directed mutagenesis.
- PAMP polypeptides produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins and PAMP mutants may be used as an immunogen to generate antibodies that recognize the PAMP polypeptide.
- Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
- Such an antibody is preferably specific for human PAMP, PAMP originating from other species, or for post-translationally modified (e.g. phosphorylated, glycosylated) PAMP.
- PAMP polypeptide or derivative or analog thereof various procedures known in the art may be used for the production of polyclonal antibodies to PAMP polypeptide or derivative or analog thereof.
- various host animals can be immunized by injection with the PAMP polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
- the PAMP polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
- BSA bovine serum albumin
- KLH keyhole limpet hemocyanin
- adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
- Antisera may be collected at a chosen time point after immunization, and purified as desired. For preparation of monoclonal antibodies directed toward the
- PAMP polypeptide, or fragment, analog, or derivative thereof any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497, 1975), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983; Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
- Such human or humanized chimeric antibodies are preferred for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogenic antibodies to induce an immune response, in particular an allergic response, themselves.
- Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques.
- such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule, the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papam and a reducing agent
- screening for the desired antibody can be accomplished by techniques known in the art, e g , radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e g , gel aggaggagg
- the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the PAMP polypeptide, e g , for Western blotting, imaging PAMP polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc using any of the detection techniques mentioned above or known in the art
- Such antibodies can be used to identify proteins that interact with PAMP, and to detect conformational or structural changes in PAMP
- antibodies that agonize or antagonize the activity of PAMP polypeptide can be generated They can also be used to regulate or inhibit PAMP activity intracellular, / e , the invention contemplates an intracellular antibody (intrabody), e g , single chain Fv antibodies (see generally, Chen, Mol Med Today, 3 160-167, 1997, Spitz et al , Anticancer Res , 16 3415-3422, 1996, Indolfi et al , Nat Med , 2 634-635, 1996, Kijima et al , Pharmacol Ther , 68 247-267, 1995)
- intracellular antibody e g
- single chain Fv antibodies see generally, Chen, Mol Med Today, 3 160-167, 1997, Spitz et al , Anticancer Res , 16 3415-3422, 1996, Indolfi et al , Nat Med , 2 634-635, 1996, Kijima et al , Pharmacol Ther , 68 247-267,
- the nucleotide sequence and the protein sequence and the putative biological activity of PAMP or PAMP mutants can all be used for the purposes of diagnosis of individuals who are at- ⁇ sk for, or who actually have, a variety of neurodegenerative diseases (including Alzheimer's disease, Lewy body variant, Parkinson's disease-dementia complex, amyotrophic lateral sclerosis etc ), neuropsychiatric diseases (schizophrenia, depression, mild cognitive impairment, benign senescent forgetfulness, age-associated memory loss, etc ), developmental disorders associated with defects in intracellular signal transduction mediated by factors such as Notch, Delta, Wingless, etc , and neoplasms (e g , bowel cancer, etc ) associated with abnormalities of PS1/PAMP/PS2 mediated regulation of cell death pathways
- These diagnostic entities can be used by searching for alterations in the nucleotide sequence of PAMP, in the transc ⁇ ptional activity of PAMP, in the protein level as monitored by immunological means (e g , EL
- PAMP Screening Assays Identification and isolation of PAMP provides for development of screening assays, particularly for high throughput screening of molecules that up- or down-regulate the activity of PAMP, e g , by permitting expression of PAMP in quantities greater than can be isolated from natural sources, or in indicator cells that are specially engineered to indicate the activity of PAMP expressed after transfection or transformation of the cells
- Any screening technique known in the art can be used to screen for PAMP agonists or antagonists
- the present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and agonize or antagonize the activity of PAMP in vivo
- natural products libraries can be screened using assays of the invention for molecules that agonize or antagonize PAMP activity
- synthetic libraries (Needels et al , Proc Natl Acad Sci USA 90 10700-4, 1993, Ohlmeyer et al , Proc Natl Acad Sci USA 90 10922-10926, 1993, Lam et al , International Patent Publ No WO 92/00252, Kocis et al , International Patent Publ No WO 9428028) and the like can be used to screen for PAMP ligands according to the present invention
- Sequences for PAMP or PAMP mutants can also be used to identify proteins which interact with PAMP either in concert with PS1 and PS2, or independently, using a variety of methods including co- immunoprecipitation, yeast two hybrid interaction trap assays, yeast three hybrid interaction trap assays, chemical cross-linking and co-precipitation studies, etc. These and other methods are described more fully in co-pending and commonly assigned U.S. Application Serial No. 08/888,077, filed July 3, " 1997, and 09/227,725, filed January 8, 1999, both of which are incorporated herein by reference. Identification of these interacting partners will then lead to their use to further delineate the biochemical pathways leading to the above-mentioned diseases.
- the structure and topology of these domains can all be used as a basis for the rational design of pharmaceuticals (small molecule conventional drugs or novel carbohydrate, lipid, DNA/RNA or protein-based high molecular weight biological compounds) to modulate (increase or decrease) the activity of PAMP and/or the PAMP PS1/PS2 complex, and/or the activity of the PAMP/other protein complexes.
- pharmaceuticals small molecule conventional drugs or novel carbohydrate, lipid, DNA/RNA or protein-based high molecular weight biological compounds
- spectroscopic data like nuclear magnetic resonance, circular dichroism, and other physical-chemical structural data, or crystallographic data, or both.
- Drug candidates generated using a rational drug design program can then be applied for the treatment and/or prevention of the above-mentioned diseases, and can be administered through a variety of means including: as conventional small molecules through enteral or pare ⁇ teral routes; via inclusion in liposome vehicles; through infusion in pumps inserted into various organs (e.g., via Omaya pumps inserted into the cerebral ventricles); via the transplantation of genetically-modified cells expressing recombinant genes; or via the use of biological vectors (e.g., retrovirus, adenovirus, adeno-associated virus, Lentivirus, or herpes simplex virus-based vectors) which allow targeted expression of appropriately modified gene products in selected cell types.
- biological vectors e.g., retrovirus, adenovirus, adeno-associated virus, Lentivirus, or herpes simplex virus-based vectors
- the recombinant proteins described above may be the wild-type PAMP, a genetically-modified PAMP, a wild-type PS1/PS2, a genetically-modified PS1/PS2, or a specially-designed protein or peptide which is designed to interact with either the functional domains of PAMP (or the PAMP/PS1/PS2/other protein complex) or to interact with the domains which modulate the activity of the functional domains of PAMP.
- the PAMP nucleotide sequence can be used to make cell-free systems, transfected cell lines, and animal models (invertebrate or vertebrate) of the neurodegenerative and other diseases outlined above. These animal and cell models may involve over-expression of all or part of PAMP or PAMP mutants, e.g., as mini-gene cDNA transgene constructs under the regulation of suitable promoter elements carried in vectors such as cos-Tet for transgenic mice and pcDNA (Invitrogen, California) in transfected cell lines.
- Animal and cellular models can also be generated by via homologous recombination mediated targeting of the endogenous gene to create artificially mutant sequences (knock-in gene targeting); or loss of function mutations (knock-out gene targeting); by translocation of P-elements; and by chemical mutagenesis.
- Animal, cellular and cell-free model systems can be used for a variety of purposes including the discovery of diagnostics and therapeutics for this disease.
- a mammal in which two or more genes have been knocked out or knocked in, or both.
- Such mammals can be generated by repeating the procedures set forth herein for generating each knockout construct, or by breeding to mammals, each with a single gene knocked out, to each other, and screening for those with the double knockout genotype.
- Regulated knockout animals can be prepared using various systems, such as the tet-repressor system (see US Patent No 5,654,168) or the Cre-Lox system (see US Patents No 4,959,317 and No 5,801 ,030)
- Transgenic mammals can be prepared for evaluating the molecular mechanisms of PAMP, and particularly human PAMP/PS1 or PAMP/PS2 function Such mammals provide excellent models for screening or testing drug candidates It is possible to evaluate compounds or diseases on "knockout" animals, e g , to identify a compound that can compensate for a defect in PAMP activity Alternatively, PAMP (or mutant PAMP), alone or in combination with ⁇ APP, PS1 , and/or PS2, (double or triple transgenics) "knock-in" mammals can be prepared for evaluating the molecular biology of this system in greater detail than is possible with human subjects Both technologies permit manipulation of single units of genetic information in their natural position in a cell genome and to examine the results of that manipulation in the background of a terminally differentiated organism These animals can be evaluated for levels of mRNA or protein expression, processing of ⁇ APP, or development of a condition indicative of inappropriate gene expression, e g , Notch phenotype or another phenotype as set forth above, or neurodegeneration, including cognitive deficits, learning
- Transgenic animal systems have been developed Mice, rats, hamsters, and other rodents are popular, particularly for drug testing, because large numbers of transgenic animals can be bred economically and rapidly Larger animals, including sheep, goats, pigs, and cows, have been made transgenic as well Transgenic D melanogaster and C elegans can also be made and, using known genetic methods, may offer the ability to identify upstream and downstream modifiers of a PAMP phenotype
- Transgenic animals can also be prepared by introducing the transgene on a vector, such animals, which are not modified in the germ line and are only transiently transgenic, naturally cannot pass along the genetic information to their progeny
- transgenic animals are created in which (i) a human PAMP, or a mutant human PAMP, is stably inserted into the genome of the transgenic animal, and/or (n) the endogenous PAMP genes are inactivated and replaced with their human counterparts See, e g , Coffman, Semm Nephrol 17 404, 1997, Esther et al , Lab Invest 74 953, 1996, Murakami et al , Blood Press Suppl 2 36, 1996 Such animals can be treated with candidate compounds and monitored for the effects of such drugs on PAMP cavity
- the present invention provides for treatment of such deficits either with a drug discovered using a screening assay or transgenic animal model, or both, as set forth above, or by replacing a defective PAMP gene with a functional gene by gene therapy
- a gene encoding PAMP, a PAMP mutant, or alternatively a negative regulator of PAMP such as an antisense nucleic acid, intracellular antibody (intrabody), or dominant negative PAMP (which may be truncated), can be introduced in vivo, ex vivo, or in vitro using a viral or a non-viral vector, e g , as discussed above
- a viral or a non-viral vector e g
- Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995
- an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e g , adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells
- immunosuppressive cytokmes such as interleukm 12 (IL-12), interferon- ⁇ (IFN ⁇ ), or ant ⁇ -CD4 antibody
- IL-12 interleukm 12
- IFN ⁇ interferon- ⁇
- ant ⁇ -CD4 antibody can be administered to block humoral or cellular immune responses to the viral vectors (see, e g , Wilson, Nature Medicine, 1995)
- a viral vector that is engineered to express a minimal number of antigens
- Herpes virus vectors Because herpes virus is trophic for cells of the nervous system (neural cells), it is an attractive vector for delivery of function PAMP genes Va ⁇ ous defective (non-replicating, and thus non- mfectious) herpes virus vectors have been described, such as a defective herpes virus 1 (HSV1 )
- Adenovirus vectors are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types in vivo, and has been used extensively in gene therapy protocols, including for targeting genes to neural cells
- Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example Mav1 , Beard et al , Virology 75 (1990) 81 ), ovine, porcine, avian, and simian (example SAV) origin
- the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2
- Adeno-associated viruses are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies
- the AAV genome has been cloned, sequenced and characterized
- the use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see WO 91/18088, WO 93/09239, US 4,797,368, US 5,139,941 , EP 488 528)
- the replication defective recombinant AAVs according to the invention can be prepared by co-transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and
- Retrovirus vectors In another embodiment the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., 1988, J. Virol. 62:1120; Temin et al., U.S. Patent No. 5,124,263; EP 453242, EP178220; Bernstein et al. Genet. Eng.
- the retroviruses are integrating viruses which infect dividing cells.
- the retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and et?v).
- the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest.
- These vectors can be constructed from different types of retrovirus, such as MoMuLV ("murine Moloney leukemia virus”), MEV (“murine Moloney sarcoma virus”), HaSV ("Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus.
- Suitable packaging cell lines have been described in the prior art, in particular the cell line PA317 (US 4,861 ,719); the PsiCRIP cell line (WO 90/02806) and the GP+envAm-12 cell line (WO 89/07150).
- the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptionai activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender et al., J. Virol. 61 :1639, 1987).
- Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.
- Retrovirus vectors can also be introduced by recombinant DNA viruses, which permits one cycle of retroviral replication and amplifies transfection efficiency (see WO 95/22617, WO 95/26411 , WO 96/39036, WO 97/19182).
- Lentivirus vectors are can be used as agents for the direct delivery and sustained expression of a transgene in several tissue types, including brain, retina, muscle, liver and blood.
- the vectors can efficiently transduce dividing and non-dividing cells in these tissues, and maintain long-term expression of the gene of interest
- Lentiviral packaging cell lines are available and known generally in the art They facilitate the production of high-titer lentivirus vectors for gene therapy
- An example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 106 lU/ml for at least 3 to 4 days (Kaf ⁇ , et al , J Virol , 73 576-584, 1999
- Non-viral vectors A vector can be introduced in vivo in a non- viral vector, e g , by hpofection, with other transfection facilitating agents (peptides, polymers, etc ), or as naked DNA Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection, with targeting in some instances (Feigner, et al , Proc Natl Acad Sci U S A 84 7413-7417, 1987, Feigner and Rmgold, Science 337 387-388, 1989, see Mackey, et al , Proc Natl Acad Sci U S A 85 8027-8031 , 1988, Ulmer et al , Science 259 1745- 1748, 1993)
- Useful hpid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U S Patent No 5,459,127 Other molecules are also useful for facilitating transfection of a nu
- EXAMPLE 1 A novel PAMP that mediates ⁇ APP processing
- HEK293 cells were transiently transfected with PAMP cDNA (SEQ ID NO 13) tagged at the 3'-end with a V5-ep ⁇ tope encoded from the pcDNA6 vector
- the conditioned media were collected 20 hr after transient transfection with PAMP (or with empty vector), and the A ⁇ and A ⁇ 42 levels were measured by ELISA (Zhang L, et al , J Biol Chem 1999, 274 8966-8972 )
- ELISA Zet L, et al , J Biol Chem 1999, 274 8966-8972
- the use of ant ⁇ -V5 Invitrogen, CA
- enhanced chemiluminescence Amersham
- detected a V5- immunoreactive band of ⁇ 110 kDa which was reduced to ⁇ 80 kDa following Endo H digestion (equivalent to the size predicted from the PAMP ammo acid sequence), confirming the predicted glycosylation of PAMP
- the PAMP gene Chromosomal locations and genetic map positions of the murine and human PAMPS were obtained from public genetic and transc ⁇ ptional maps (www ncbi nlm nih gov)
- the gene encoding PAMP is located on human chromosome 1 near the genetic markers D1 S1595 and D1 S2844
- the 5'- end of the PAMP gene is embedded in the 5'- end of the coatmer gene encoded on the opposite strand
- the human PAMP gene is close to a cluster of markers which have yielded positive, but sub-significant evidence for linkage to or association with Alzheimer Disease in two independent genome wide surveys (Kehoe P, et al Hum Mol Genet 1999, 8 237-245)
- the murine PAMP maps within a 700 Kb interval of murine chromosome 1 which contains the gene defect associated with Looptail phenotype in mice (Underhill DA, et al , Genomics 1999, 55 185-193) Mice heterozygous for Looptail show
- PAMP corresponds to the aph-2 gene Mutations in aph-2 have been identified in a screen for mutants with phenotypes identical to embryonic mutant phenotypes caused by loss of glp-1 activity, / e , lack of an anterior pharynx, e g cDNA clone
- Double stranded RNA interference (RNAi) confirmed the mutant phenotype of aph-2
- Sense and antisense RNA were transcribed in vitro from PCR product amplified from the phage yk477b8 After annealing equal quantities of sense and antisense products, the d
- the PS1 -L392V mutation is a pathogenic mutation associated with familial AD (Sher ⁇ ngton R, et al , Nature 1995, 375 754-760) and with increased secretion of A ⁇ 2 (Scheuner D, et al Nature Med 1996, 2 864-870, Citron M, et al Nature Med 1997, 3 67-72)
- the PS1 -D385A mutation is a loss of function mutation associated with inhibition of PS1 endoproteolysis and a decrease
- Presenilin binding domains of PAMP In transiently transfected cells (in which the 7-10 kDa C-termmal of PAMP can be detected), ant ⁇ -PS1 immunoprecipitation products contain both full length PAMP and the -7-10 kDa C-termmal PAMP fragments Similarly, in these cells, immunoprecipitation with antibodies to the C-termmus of ⁇ APP (Ab369) also gives C-termmal PAMP epitopes The TM domain of PAMP is not highly conserved in evolution These results suggest that the C99-/C83- ⁇ APP and PS1/PS2-b ⁇ nd ⁇ ng doma ⁇ n(s) of PAMP are in the TM domain or C-terminus
- PAMP is a major stoichiomet c component of the presenilin complexes.
- PAMP selectively interacts only with C-terminal derivatives of ⁇ APP which are substrates for ⁇ -secretase cleavage, and this interaction is modulated by PS ⁇ mutations in a way which reflects the functional consequences of these PS1 mutations.
- inhibition of PAMP expression in C. elegans leads to a disease phenotype likely to be in the glp/Notch signaling pathway.
- TM PAMP S 683A in the TM domain
- C3D PAMP ⁇ 63o-66 ⁇ in the conserved region adjacent to the TM domain
- PAMP wt normal/wild type PAMP
- LacZ unrelated protein
- This domain contains no obvious functional motifs (e.g., for glycosylation etc.), nor does it have significant sequence homology to other known proteins. Consequently, the three functionally active PAMP mutations either affect a presenilin-binding domain in PAMP, or affect a specific regulatory domain of PAMP which modulates both direct binding of PAMP to PS1 and the subsequent ⁇ - secretase-mediated cleavage of PAMP-bound C99- and C83- ⁇ APP stubs.
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US173826P | 1999-12-30 | ||
PCT/CA2000/000354 WO2000060069A1 (en) | 1999-04-01 | 2000-04-03 | A presenilin associated membrane protein and uses thereof |
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AU2002361682A1 (en) * | 2001-12-14 | 2003-06-30 | Elizabeth Kopp | Innate immune system-directed vaccines |
US8420102B2 (en) | 2006-03-07 | 2013-04-16 | Vaxinnate Corporation | Compositions that include hemagglutinin |
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