WO2010065953A1 - Gènes alpha de récepteur de lymphocyte t qui prédisposent les voies respiratoires à la colonisation bactérienne - Google Patents

Gènes alpha de récepteur de lymphocyte t qui prédisposent les voies respiratoires à la colonisation bactérienne Download PDF

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WO2010065953A1
WO2010065953A1 PCT/US2009/066980 US2009066980W WO2010065953A1 WO 2010065953 A1 WO2010065953 A1 WO 2010065953A1 US 2009066980 W US2009066980 W US 2009066980W WO 2010065953 A1 WO2010065953 A1 WO 2010065953A1
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asthma
nucleic acid
cells
association
bacterial colonization
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PCT/US2009/066980
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English (en)
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Hans Bisgaard
Patrick M.A. Sleiman
Hakon Hakonarson
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The Children's Hospital Of Philadelphia
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • This invention relates to the fields of airway disease, immunity, bacterial colonization, and genetic testing. More specifically, the invention provides compositions and methods for the diagnosis and treatment of asthma and other allergic conditions.
  • Asthma is a heterogeneous multifactorial disease that manifests as episodes of wheezing, coughing and breathlessness. Airway inflammation together with bronchial hyperresponsiveness, atopy, and elevated IgE and impaired adrenoceptor-mediated airway relaxation are characteristic features of bronchial asthma. Multiple genetic and environmental factors are known to modulate clinical expression of the disease and its associated phenotypes (2). However, the genetic susceptibility factors underpinning what is the most common chronic childhood disease remain largely unknown. Asthma is a major health problem in children and an immense economic burden on the health care systems both in the US and the rest of the world.
  • GWA is the most robust approach to gene variant discovery (4). However, unlike the discovery of multiple variants underlying type-2 diabetes or height (5)(6), only one locus has been uncovered (3), suggesting that either substantially larger numbers of subjects need to be studied or individuals who might possess a higher proportion of 'genetic' disease either through lower age of onset or increased severity need to be ascertained.
  • compositions and methods are provided for identifying children exhibiting increased levels of bacterial colonization in their airways and therefore at an increased risk for developing asthma.
  • An exemplary method entails detecting the presence of a single nucleotide polymorphism on chromosome 14 in a TCRa encoding polynucleotide wherein if the single nucleotide polymorphism is present, the patient has an a propensity for enhanced bacterial colonization of the airway and a subsequent increased risk for developing asthma.
  • Exemplary single nucleotide polymorphisms associated with the development of asthma include, without limitation, those disclosed in Table 1 hereinbelow.
  • the methods of the invention can include alternative means for detecting the disclosed polymorphisms.
  • methods of detection can further comprises processes such as specific hybridization, measurement of allele size, restriction fragment length polymorphism analysis, allele-specific hybridization analysis, single base primer extension reaction, and sequencing of an amplified polynucleotide.
  • the polymorphism is on chromosome 14 and is provided in Table 1.
  • nucleic acid molecules useful for amplifying the nucleic acids encoding the single nucleotide polymorphisms disclosed herein are provided.
  • solid supports comprising suitable nucleic acid targets to facilitate detection of such SNPS in patient samples.
  • a suitable solid support for this process includes a microarray.
  • the invention also encompasses screening methods to identify agents which differentially modulate TCRa action or function in the SNP containing cells described herein.
  • An exemplary method entails providing cells comprising at least one of the SNPs disclosed in Table 1 ; providing cells which express these gene(s) which lack the cognate polymorphisms (step b); contacting each cell type with a test agent and analyzing whether said agent alters T cell function and/or signaling , agents so identified are also within the scope of the invention.
  • transgenic mice comprising the SNP containing nucleic acid molecules described herein. Such mice provide a superior in vivo screening tool to identify agents which modulate the progression and development of asthma.
  • Figure 1 shows a linkage disequilibrium heatmap.
  • Figure 2 shows multiple SNPs and their association with FEV 05>
  • Figure 3 shows multiple SNPs and their association with PD 15 TcO 2 .
  • Figure 4 shows multiple SNPs and their association with PD 20 metacholine.
  • FIG. 5 shows multiple SNPs and their association with FEVi.
  • Figure 6 shows multiple SNPs and their association with FEVi/FVC.
  • Figure 7 shows multiple SNPs and their association with MMEF.
  • Figure 8 shows multiple SNPs and their association with sRaw profiles.
  • Figure 9 shows multiple SNPs and their association with eosinophilocytes at age 6 years.
  • Figure 10 shows multiple SNPs and their association with asthma status at age 6 years.
  • Figure 11 shows multiple SNPs and their association with whez categorization.
  • Figure 12 shows multiple SNPs and their association with bacterial colonization.
  • Figure 13 shows multiple SNPs and their association with JJJ.
  • Figure 14 shows multiple SNPs and their association with J45.
  • Figure 15 shows multiple SNPs and their association with exacerbation.
  • Figure 16 shows multiple SNPs and their association with exacerbation incidence.
  • Figure 17 shows multiple SNPs and their association with eczema.
  • Figure 18 shows multiple SNPs and their association with asthma related events.
  • Figure 19 shows haplotype association analysis performed against colonization phenotype. Significant association detected with haplotypes formed by block 1 which includes the most highly associated SNP rs227870. A single haplotype (the second most common in Europeans) is highly associated with the colonization phenotype.
  • Asthma is the most common chronic disease in children across all developed countries. The incidence of the disease is currently increasing world-wide. Family and twin studies strongly implicate genetic factors in an individual's risk of developing of asthma. Despite this, it has proven difficult to isolate disease genes that confer susceptibility to this disease using classical candidate gene and linkage approaches, with the notable exception of linkages to the HLA loci on chr 5q3 l-q33 and 6p21. Over the last two years, genome-wide association (GWA) studies have become feasible, where modern high-throughput SNP genotyping technologies can be applied to large and comprehensively phenotyped patient cohorts. Such approaches have enabled scientists to robustly associate specific variants with many complex diseases, including age-related macular degeneration, type 2 diabetes, breast cancer and IBD.
  • GWA genome-wide association
  • This invention pertains to the discovery of another novel variant that confers a predisposition to bacterial colonization of the airways of newborns and infants and associates robustly with the development of asthma in later life. Association results are presented that for the first time make the link between bacterial colonization of newborns and infants and the development of asthma in later life. This discovery will facilitate identifying those patients most likely to develop asthma and also provides new targets for the design agents and compositions useful for the treatment of asthma.
  • a or “an” entity refers to one or more of that entity; for example, "a cDNA” refers to one or more cDNA or at least one cDNA.
  • a cDNA refers to one or more cDNA or at least one cDNA.
  • the terms “a” or “an,” “one or more” and “at least one” can be used interchangeably herein.
  • the terms “comprising,” “including,” and “having” can be used interchangeably.
  • a compound “selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds.
  • an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, Aisolated ⁇ and Abiologically pure ⁇ do not necessarily reflect the extent to which the compound has been purified.
  • An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical
  • Enhanced bacterial colonization/asthma-associated SNP or specific marker is a SNP or marker which is associated with an increased or decreased predisposition to bacterial colonization, such increased colonization conferring an risk of developing asthma not found normal patients who do not have this disease.
  • markers may include but are not limited to nucleic acids, proteins encoded thereby, or other small molecules.
  • SNP single nucleotide polymorphism
  • genetic alteration refers to a change from the wild-type or reference sequence of one or more nucleic acid molecules. Genetic alterations include without limitation, base pair substitutions, additions and deletions of at least one nucleotide from a nucleic acid molecule of known sequence.
  • solid matrix refers to any format, such as beads, microparticles, a microarray, the surface of a microtitration well or a test tube, a dipstick or a filter.
  • the material of the matrix may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran or agarose.
  • phrases "consisting essentially of when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO:.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.
  • Target nucleic acid refers to a previously defined region of a nucleic acid present in a complex nucleic acid mixture wherein the defined wild-type region contains at least one known nucleotide variation which may or may not be associated with asthma.
  • the nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually.
  • the nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer.
  • the term "isolated nucleic acid” is sometimes employed. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived.
  • the "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote.
  • An "isolated nucleic acid molecule” may also comprise a cDNA molecule.
  • An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a recombinant nucleic acid molecule.
  • isolated nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure” form.
  • enriched in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that “enriched” does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • nucleotide sequence be in purified form.
  • purified in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/ml).
  • Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity.
  • the claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA.
  • the cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library.
  • the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 " 6 -fold purification of the native message.
  • purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest.
  • complementary describes two nucleotides that can form multiple favorable interactions with one another.
  • adenine is complementary to thymine as they can form two hydrogen bonds.
  • guanine and cytosine are complementary since they can form three hydrogen bonds.
  • a "complement" of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine.
  • the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.
  • “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • specific hybridization can refer to a sequence which hybridizes to any asthma specific marker nucleic acid, but does not hybridize to other nucleotides.
  • polynucleotide which "specifically hybridizes" may hybridize only to a TCRa specific marker, such as the TCRa-specific markers shown in the Tables contained herein. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.
  • the T n is 57 " C.
  • the T m of a DNA duplex decreases by 1 - 1.5 " C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 " C.
  • the stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25 0 C below the calculated T m of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20 0 C below the T m of the hybrid.
  • a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42 0 C, and washed in 2X SSC and 0.5% SDS at 55°C for 15 minutes.
  • a high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in IX SSC and 0.5% SDS at 65°C for 15 minutes.
  • a very high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in 0.1X SSC and 0.5% SDS at 65°C for 15 minutes.
  • oligonucleotide is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide. Oligonucleotides, which include probes and primers, can be any length from 3 nucleotides to the full length of the nucleic acid molecule, and explicitly include every possible number of contiguous nucleic acids from 3 through the full length of the polynucleotide. Preferably, oligonucleotides are at least about 10 nucleotides in length, more preferably at least 15 nucleotides in length, more preferably at least about 20 nucleotides in length.
  • probe refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5 Or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
  • primer refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.
  • suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH
  • the primer may be extended at its 3 * terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.
  • the primer may vary in length depending on the particular conditions and requirement of the application.
  • the oligonucleotide primer is typically 15-25 or more nucleotides in length.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template.
  • a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer.
  • non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • siRNA refers to a molecule involved in the RNA interference process for a sequence-specific post-transcriptional gene silencing or gene knockdown by providing small interfering RNAs (siRNAs) that has homology with the sequence of the targeted gene.
  • small interfering RNAs can be synthesized in vitro or generated by ribonuclease III cleavage from longer dsRNA and are the mediators of sequence-specific mRNA degradation.
  • the siRNA of the invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • the siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • Commercial suppliers of synthetic RNA molecules or synthesis reagents include Applied Biosystems (Foster City, CA, USA), Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).
  • Specific siRNA constructs for inhibiting DENN/D1B mRNA for example, may be between 15-35 nucleotides in length, and more typically about 21 nucleotides in length.
  • vector relates to a single or double stranded circular nucleic acid molecule that can be infected, transfected or transformed into cells and replicate independently or within the host cell genome.
  • a circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes.
  • restriction enzymes An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that are targeted by restriction enzymes are readily available to those skilled in the art, and include any replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
  • a nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
  • transformation transformation
  • transfection transduction
  • Atransduction ⁇ refers to methods of inserting a nucleic acid and/or expression construct into a cell or host organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest, microinjection, PEG-fusion, and the like.
  • promoter element describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA.
  • the promoter element of the present invention precedes the 5 1 end of the marker nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide.
  • nucleic acid vector can contain nucleic acid elements other than the promoter element and the asthma specific marker encoding nucleic acid.
  • nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, localization signals, or signals useful for polypeptide purification.
  • a “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, plastid, phage or virus, that is capable of replication largely under its own control.
  • a replicon may be either RNA or DNA and may be single or double stranded.
  • an "expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • transcriptional and translational control sequences such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • reporter As used herein, the terms “reporter,” “reporter system”, “reporter gene,” or “reporter gene product” shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods.
  • the nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product.
  • the required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.
  • the introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism.
  • the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid.
  • the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism.
  • the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
  • selectable marker gene refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.
  • recombinant organism or “transgenic organism” refer to organisms which have a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art.
  • organism relates to any living being comprised of a least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. Therefore, the phrase "a recombinant organism” encompasses a recombinant cell, as well as eukaryotic and prokaryotic organism.
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form. "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.
  • a “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules.
  • specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair comprises nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.
  • Sample or “patient sample” or “biological sample” generally refers to a sample which may be tested for a particular molecule, preferably an bacterial colonization/asthma specific marker molecule, such as a marker shown in the tables provided below.
  • Samples may include but are not limited to cells, body fluids, including blood, serum, plasma, urine, saliva, tears, pleural fluid and the like.
  • agent and “test compound” are used interchangeably herein and denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • Bio macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide/DNA complexes, and any nucleic acid based molecule which exhibits the capacity to modulate the activity of the SNP containing nucleic acids described herein or their encoded proteins. Agents are evaluated for potential biological activity by inclusion in screening assays described hereinbelow.
  • Bacterial colonization/asthma-related-SNP containing nucleic acids may be used for a variety of purposes in accordance with the present invention.
  • Enhanced bacterial colonization/asthma-associated SNP containing DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of the same in biological samples.
  • Methods in which the specific marker nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).
  • assays for detecting enhanced bacterial colonization/asthma-associated SNPs or the proteins encoded thereby may be conducted on any type of biological sample, including but not limited to body fluids (including blood, urine, serum, gastric lavage), any type of cell (such as brain cells, white blood cells, mononuclear cells) or body tissue.
  • body fluids including blood, urine, serum, gastric lavage
  • any type of cell such as brain cells, white blood cells, mononuclear cells
  • enhanced bacterial colonization/asthma-associated SNP containing nucleic acids, vectors expressing the same, SNP containing marker proteins and anti-SNP specific marker antibodies of the invention can be used to detect such SNPs in body tissue, cells, or fluid, and alter SNP containing marker protein expression for purposes of assessing the genetic and protein interactions involved in the predisposition to bacterial colonization and the subsequent development of asthma.
  • the SNP containing nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the templates as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are becoming increasingly important in the art.
  • new detection technologies can overcome this limitation and enable analysis of small samples containing as little as l ⁇ g of total RNA.
  • RLS Resonance Light Scattering
  • PWG planar wave guide technology
  • any of the aforementioned techniques may be used to detect or quantify enhanced bacterial colonization/asthma-associated SNP marker expression and accordingly, diagnose an increased risk of developing asthma.
  • kits which may contain a enhanced bacterial colonization/asthma-associated SNP specific marker polynucleotide or one or more such markers immobilized on a Gene Chip, an oligonucleotide, a polypeptide, a peptide, an antibody, a label, marker, or reporter, a pharmaceutically acceptable carrier, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, an assay substrate, or any combination thereof.
  • a kit which may contain a enhanced bacterial colonization/asthma-associated SNP specific marker polynucleotide or one or more such markers immobilized on a Gene Chip, an oligonucleotide, a polypeptide, a peptide, an antibody, a label, marker, or reporter, a pharmaceutically acceptable carrier, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, an assay substrate, or any combination thereof.
  • Chromosome 14 contains TCRa protein coding regions which provide suitable targets for the rational design of therapeutic agents which modulate their activity. Small peptide molecules corresponding to these regions may be used to advantage in the design of therapeutic agents which effectively modulate the activity of the encoded proteins.
  • Molecular modeling should facilitate the identification of specific organic molecules with capacity to bind to the active site of the proteins encoded by the SNP containing nucleic acids based on conformation or key amino acid residues required for function.
  • a combinatorial chemistry approach will be used to identify molecules with greatest activity and then iterations of these molecules will be developed for further cycles of screening.
  • the polypeptides or fragments employed in drug screening assays may either be free in solution, affixed to a solid support or within a cell.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may determine, for example, formation of complexes between the polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between the polypeptide or fragment and a known substrate is interfered with by the agent being tested.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity for the encoded polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different, small peptide test compounds, such as those described above, are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the target polypeptide and washed. Bound polypeptide is then detected by methods well known in the art.
  • a further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as T cells) which have a nonfunctional or altered bacterial colonization/asthma associated TCRa gene. These host cell lines or cells therefore encode TCRa altered at the polypeptide level.
  • the host cell lines or cells are exposed to known asthma triggers, including bacterial toxins, allergens, viral particles, or other activators and then grown in the presence of a test compound to determine if the compound is capable of regulating T cell signialling or function in altered cells.
  • the enhanced bacterial colonization/asthma- associated SNP containing TCRa encoding DNA molecules may be introduced singly into such host cells or in combination to assess the phenotype of cells conferred by such expression.
  • Suitable vectors for use in practicing the invention include prokaryotic vectors such as the pNH vectors (Stratagene Inc., 11099 N. Torrey Pines Rd., La Jolla, Calif. 92037), pET vectors (Novogen Inc., 565 Science Dr., Madison, Wis. 53711) and the pGEX vectors (Pharmacia LKB Biotechnology Inc., Piscataway, N.J. 08854).
  • Examples of eukaryotic vectors useful in practicing the present invention include the vectors pRc/CMV, pRc/RSV, and pREP (Invitrogen, 11588 Sorrento Valley Rd., San Diego, Calif.
  • pcDNA3.1/V5&His Invitrogen
  • baculovirus vectors such as pVL1392, pVL1393, or pAC360 (Invitrogen)
  • yeast vectors such as YRP17, YIP5, and YEP24 (New England Biolabs, Beverly, Mass.), as well as pRS403 and pRS413 Stratagene Inc.
  • Picchia vectors such as pHIL-Dl (Phillips Petroleum Co., Bartlesville, OkIa. 74004)
  • retroviral vectors such as PLNCX and pLPCX (Clontech)
  • adenoviral and adeno-associated viral vectors adenoviral and adeno-associated viral vectors.
  • Promoters for use in expression vectors of this invention include promoters that are operable in prokaryotic or eukaryotic cells. Promoters that are operable in prokaryotic cells include lactose (lac) control elements, bacteriophage lambda (pL) control elements, arabinose control elements, tryptophan (trp) control elements, bacteriophage T7 control elements, and hybrids thereof.
  • lac lactose
  • pL bacteriophage lambda
  • trp tryptophan
  • Promoters that are operable in eukaryotic cells include Epstein Barr virus promoters, adenovirus promoters, SV40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus (CMV) promoters, baculovirus promoters such as AcMNPV polyhedrin promoter, Picchia promoters such as the alcohol oxidase promoter, and Saccharomyces promoters such as the gal4 inducible promoter and the PGK constitutive promoter.
  • a vector of this invention may contain any one of a number of various markers facilitating the selection of a transformed host cell. Such markers include genes associated with temperature sensitivity, drug resistance, or enzymes associated with phenotypic characteristics of the host organisms.
  • Host cells expressing the enhanced bacterial colonization/asthma-associated SNPs of the present invention or functional fragments thereof provide a system in which to screen potential compounds or agents for the ability to modulate bacterial colonization and/or the development of asthma.
  • the nucleic acid molecules of the invention may be used to create recombinant cell lines for use in assays to identify agents which modulate aspects of airway bacterial colonization and the subsequent cellular signaling associated with the development of asthma and aberrant bronchoconstriction.
  • methods to screen for compounds capable of modulating the function of proteins encoded by SNP containing nucleic acids are also provided herein.
  • Another approach entails the use of phage display libraries engineered to express fragment of the polypeptides encoded by the SNP containing nucleic acids on the phage surface. Such libraries are then contacted with a combinatorial chemical library under conditions wherein binding affinity between the expressed peptide and the components of the chemical library may be detected.
  • US Patents 6,057,098 and 5,965,456 provide methods and apparatus for performing such assays.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, (1991) Bio/Technology 9:19-21.
  • the three-dimensional structure of a protein of interest or, for example, of the protein-substrate complex is solved by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches.
  • peptides may be analyzed by an alanine scan (Wells, (1991) Meth. Enzym. 202:390- 411). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • anti-idiotypic antibodies As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original molecule.
  • the anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.
  • drugs which have, e.g., improved polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of polypeptide activity.
  • SNP containing nucleic acid sequences described herein sufficient amounts of the encoded polypeptide may be made available to perform such analytical studies as x-ray crystallography.
  • the knowledge of the protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
  • the availability of enhanced bacterial colonization/asthma- associated SNP containing nucleic acids enables the production of strains of laboratory mice carrying the enhanced bacterial colonization/asthma-associated SNPs of the invention.
  • Transgenic mice expressing the enhanced bacterial colonization/asthma-associated SNP of the invention provide a model system in which to examine the role of the altered TCRa protein encoded by the SNP containing nucleic acid in bacterial colonization mediated development and progression towards asthma.
  • Methods of introducing transgenes in laboratory mice are known to those of skill in the art. Three common methods include: 1. integration of retroviral vectors encoding the foreign gene of interest into an early embryo; 2.
  • mice provide an in vivo screening tool to study putative therapeutic drugs in a whole animal model and are encompassed by the present invention.
  • animal is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
  • a "transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus.
  • transgenic animal is not meant to encompass classical crossbreeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule.
  • This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • the term "germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring, in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.
  • the alteration of genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene. Such altered or foreign genetic information would encompass the introduction of enhanced bacterial colonization/asthma- associated SNP containing nucleotide sequences.
  • the DNA used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.
  • ES cells may be obtained from pre-implantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069).
  • Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus-mediated transduction.
  • the resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
  • the introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
  • TCRa genes and their expression products are used as insertional cassettes to selectively inactivate a wild-type gene in totipotent ES cells (such as those described above) and then generate transgenic mice.
  • the use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described, and is reviewed elsewhere (Frohman et al., (1989) Cell 56:145-147; Bradley et al., (1992) Bio/Technology 10:534-539).
  • Non-homologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with effective herpes drugs such as gancyclovir
  • HSV-TK Herpes Simplex virus thymidine kinase
  • GANC GANC
  • FIAU l-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodou- racil
  • a knock-in animal is one in which the endogenous murine gene, for example, has been replaced with human enhanced bacterial colonization/asthma-associated SNP containing TCRa gene of the invention.
  • Such knock-in animals provide an ideal model system for studying enhanced bacterial colonization and the subsequent increased risk for the development of asthma.
  • the expression of a enhanced bacterial colonization/asthma-associated SNP containing TCRa nucleic acid, fragment thereof, or an enhanced bacterial colonization/asthma-associated SNP containing TCRa fusion protein can be targeted in a "tissue specific manner" or "cell type specific manner” using a vector in which nucleic acid sequences encoding all or a portion the TCRa SNP(s) are operably linked to regulatory sequences (e.g., promoters and/or enhancers) that direct expression of the encoded protein in a particular tissue or cell type.
  • regulatory sequences e.g., promoters and/or enhancers
  • Promoters for directing tissue specific proteins are well known in the art and described herein.
  • the nucleic acid sequence encoding the enhanced bacterial colonization/asthma- associated SNP containing TCRa of the invention may be operably linked to a variety of different promoter sequences for expression in transgenic animals.
  • promoters include, but are not limited to a prion gene promoter such as hamster and mouse Prion promoter (MoPrP), described in U.S. Pat. No. 5,877,399 and in Borchelt et al., Genet. Anal. 13(6) (1996) pages 159-163; a rat neuronal specific enolase promoter, described in U.S. Pat. Nos. 5,612,486, and 5,387,742; a platelet-derived growth factor B gene promoter, described in U.S.
  • a prion gene promoter such as hamster and mouse Prion promoter (MoPrP), described in U.S. Pat. No. 5,877,399 and in Borchelt et al., Genet. Anal. 13
  • transgenic mice into which a nucleic acid containing the enhanced bacterial colonization/asthma-associated SNP or its encoded TCRa protein have been introduced are useful, for example, to develop screening methods to screen therapeutic agents to identify those capable of modulating bacterial colonization and the increased risk for the development of asthma.
  • compositions useful for treatment and diagnosis of asthma may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, aerosolized, intramuscular, and intraperitoneal routes.
  • the invention includes a method of treating asthma in a mammal.
  • An exemplary method entails administering to the mammal a pharmaceutically effective amount of siRNA which down regulates expression of the altered TCRa genes of the invention.
  • the mammal is a human.
  • the term "patient” as used herein refers to a human.
  • siRNA preparations directed at inhibiting the expression of TCRa are provided as a novel therapy to treat asthma.
  • SiRNA oligonucleotides directed to TCRa specifically hybridize with nucleic acids encoding TCRa and interfere with TCRa gene expression.
  • the siRNA can be delivered to a patient in vivo either systemically or locally with carriers, as discussed below.
  • the compositions of the invention may be used alone or in combination with other agents or genes encoding proteins to augment the efficacy of the compositions.
  • a “membrane permeant peptide sequence” refers to a peptide sequence which is able to facilitate penetration and entry of the TCRa inhibitor across the cell membrane.
  • Exemplary peptides include with out limitation, the signal sequence from Karposi fibroblast growth factor exemplified herein, the HIV tat peptide (Vives et al., J Biol. Chem., 272:16010- 16017, 1997), Nontoxic membrane translocation peptide from protamine (Park et al., FASEB J. 19(11):1555-7, 2005), CHARIOT® delivery reagent (Active Motif; US Patent 6,841,535) and the antimicrobial peptide Buforin 2.
  • siRNAs are delivered for therapeutic benefit.
  • There are several ways to administer the siRNA of the invention to in vivo to treat or ameliorate the symptoms of asthma including, but not limited to, naked siRNA delivery, siRNA conjugation and delivery, liposome carrier-mediated delivery, polymer carrier delivery, nanoparticle compositions, plasmid-based methods, and the use of viruses.
  • siRNA composition of the invention can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. This can be necessary to allow the siRNA to cross the cell membrane and escape degradation.
  • a delivery vehicle including liposomes
  • This can be necessary to allow the siRNA to cross the cell membrane and escape degradation.
  • Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, MoI. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp.
  • the frequency of administration of the siRNA to a patient will also vary depending on several factors including, but not limited to, the type and severity of the asthma to be treated, the route of administration, the age and overall health of the individual, the nature of the siRNA, and the like. It is contemplated that the frequency of administration of the siRNA to the patient may vary from about once every few months to about once a month, to about once a week, to about once per day, to about several times daily.
  • compositions that are useful in the methods of the invention may be administered systemically in parenteral, oral solid and liquid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations.
  • these pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration.
  • Such compositions may optionally contain other components, such as adjuvants, e.g., aqueous suspensions of aluminum and magnesium hydroxides, and/or other pharmaceutically acceptable carriers, such as saline.
  • nanoparticles such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer the appropriate siRNA to a patient according to the methods of the invention.
  • nanoparticles to deliver siRNAs, as well as cell membrane permeable peptide carriers that can be used are described in Crombez et al., Biochemical Society Transactions v35:p44 (2007).
  • siRNA is administered to an individual as a pharmaceutical composition comprising siRNA and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include aqueous solutions such as physiologically buffered saline, other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize the siRNA or increase the absorption of the agent.
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • carbohydrates such as glucose, sucrose or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such as ascorbic acid or glutathione
  • low molecular weight proteins or other stabilizers or excipients include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • a pharmaceutical composition comprising siRNA can be administered to a subject by various routes including, for example, orally or parenterally, such as intravenously (i.v.), intramuscularly, subcutaneously, intraorbitally, intranasally, intracapsularly, intraperitoneally (i.p.), intracisteraally, intra-tracheally (i.t.), or intra-articularly or by passive or facilitated absorption.
  • routes of administration can be used other pharmaceutically useful compounds, for example, small molecules, nucleic acid molecules, peptides, antibodies and polypeptides as discussed hereinabove.
  • a pharmaceutical composition comprising siRNA inhibitor also can be incorporated, if desired, into liposomes, microspheres, microbubbles, or other polymer matrices (Gregoriadis, Liposome Technology, VoIs. I to III, 2nd ed., CRC Press, Boca Raton FIa. (1993)).
  • Liposomes for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • the pharmaceutical preparation comprises a siRNA targeting a nucleic acid target of interest or an expression vector encoding the same.
  • Such pharmaceutical preparations can be administered to a patient for treating asthma, for example..
  • Expression vectors for the expression of siRNA molecules preferably employ a strong promoter which may be constitutive or regulated.
  • promoters are well known in the art and include, but are not limited to, RNA polymerase II promoters, the T7 RNA polymerase promoter, and the RNA polymerase III promoters U6 and Hl (see, e.g., Myslinski et al. (2001) Nucl. Acids Res., 29:2502 09).
  • a formulated siRNA composition can be a composition comprising one or more siRNA molecules or a vector encoding one or more siRNA molecules independently or in combination with a cationic lipid, a neutral lipid, and/or a polyethyleneglycol-diacylglycerol (PEG-DAG) or PEG-cholesterol (PEG-Chol) conjugate.
  • PEG-DAG polyethyleneglycol-diacylglycerol
  • PEG-Chol PEG-cholesterol
  • a lipid nanoparticle composition is a composition comprising one or more biologically active molecules independently or in combination with a cationic lipid, a neutral lipid, and/or a polyethyleneglycol-diacylglycerol (i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB) conjugate.
  • a polyethyleneglycol-diacylglycerol i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB
  • the biologically active molecule is encapsulated in the lipid nanoparticle as a result of the process of providing and aqueous solution comprising a biologically active molecule of the invention (i.e., siRNA), providing an organic solution comprising lipid nanoparticle, mixing the two solutions, incubating the solutions, dilution, ultrafiltration, resulting in concentrations suitable to produce nanoparticle compositions.
  • Nucleic acid molecules can be administered to cells by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins. (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos.
  • WO 03/47518 and WO 03/46185 poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722)
  • Cationic lipids and polymers are two classes of non- viral siRNA delivery which can form complexes with negatively charged siRNA.
  • the self-assembly PEG-ylated polycation polyethylenimine (PEI) has also been used to condense and protect siRNAs (Schiffelers et al., 2004, Nuc. Acids Res. 32: 141-110).
  • PEI polycation polyethylenimine
  • the siRNA complex can be condensed into a nanoparticle to allow efficient uptake of the siRNA through endocytosis.
  • the nucleic acid-condensing property of protamine has been combined with specific antibodies to deliver siRNAs and can be used in the invention (Song et al., 2005, Nat Biotech. 23:709-717).
  • siRNA should be administered in an effective dose.
  • the total treatment dose can be administered to a subject as a single dose or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a more prolonged period of time, for example, over the period of a day to allow administration of a daily dosage or over a longer period of time to administer a dose over a desired period of time.
  • a fractionated treatment protocol in which multiple doses are administered over a more prolonged period of time, for example, over the period of a day to allow administration of a daily dosage or over a longer period of time to administer a dose over a desired period of time.
  • the amount of siRNA required to obtain an effective dose in a subject depends on many factors, including the age, weight and general health of the subject, as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose so as to obtain an effective dose for treating an individual having asthma.
  • the effective dose of siRNA will depend on the mode of administration, and the weight of the individual being treated.
  • the dosages described herein are generally those for an average adult but can be adjusted for the treatment of children.
  • the dose will generally range from about 0.001 mg to about 1000 mg.
  • the concentration of siRNA in a particular formulation will depend on the mode and frequency of administration.
  • a given daily dosage can be administered in a single dose or in multiple doses so long as the siRNA concentration in the formulation results in the desired daily dosage.
  • One skilled in the art can adjust the amount of siRNA in the formulation to allow administration of a single dose or in multiple doses that provide the desired concentration of siRNA over a given period of time.
  • siRNA can be particularly useful when administered in combination, for example, with a conventional agent for treating such a disease.
  • a conventional agent for treating such a disease The skilled artisan would administer siRNA, alone or in combination and would monitor the effectiveness of such treatment using routine methods such as pulmonary function determination, radiologic, immunologic or, where indicated, histopathologic methods.
  • Other conventional agents for the treatment of asthma include steroid or administration of other agents that alleviate the symptoms underlying the disease.
  • Administration of the pharmaceutical preparation is preferably in an "effective amount" this being sufficient to show benefit to the individual. This amount prevents, alleviates, abates, or otherwise reduces the severity of asthma symptoms in a patient.
  • Dosage unit form refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art.
  • Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.
  • T cell receptor alpha gene predisposes to bacterial colonization and associates with asthma.
  • Novel sequences variants of TCRa have been identified which correlate with increased bacterial colonization of airways in infants resulting in an increased risk for development of asthma later in life.
  • Table 2 Coding of the genetics data according to five models of inheritance (A stands for the most frequent allele and B for the rare allele).
  • A/A A/B B/B codominant A/A A/B B/B dominant A/A A/B-B/B A/B-B/B recessive A/A-A/B A/A-A/B B/B overdominant A/A-B/B A/B A/A-B/B log-additive 0 1 2
  • the multi-SNPs association figures present the -log(/?-values) of the association between the different endpoints and the children genotypes. Univariate tests are performed SNP by SNP. The horizontal lines represent the nominal (simple dashed-line) and Bonferroni corrected (bold dashed-line)/?- value threshold.
  • Tables present the estimates (continuous variables), hazard-ratios (binary and censored data) and chi-square contributions (categorical data) for each genotype together with the levels of significance (p-values) according to the codominant, dominant and recessive genetic models.
  • Table 3 describes for each SNP the alleles, the major allele frequency, p- values of the Hardy- Weinberg equilibrium testing and the percentage of missing values. The tests of the Hardy- Weinberg equilibrium were non-significant for all SNPs but rs 17794137 for which no estimate was available since only 2 out of 3 genotypes were represented in our cohort.
  • D p(AB)-p(A)p( ⁇ ) and p(A) defined as the observed probability of allele 'A' for marker ⁇ ;
  • p(B) is defined as the observed probability of allele 'B' for marker 2;
  • p(AB) is defined as the probability of the marker allele pair 'AB'.
  • PD 15 TcO 2 measures at birth were calibrated for lifespan and weight. The association between log-transformed PD TcO 2 values and genotypes was tested. Figure 3 shows the associations of multiple SNPs with PD 15 TcO 2 .
  • Figure 4 shows multiple SNPs and their association with PD metacholine.
  • Figure 7 shows multiple SNPs and their association with MMEF.
  • Table 9 MMEF at age 6 years estimates and significance
  • Figure 8 shows multiple SNPs and their association with sRaw profiles.
  • Figure 10 shows multiple SNPs and their association with the asthma status at age 6 years
  • Figure 12 shows multiple SNPs and their association with bacterial colonization.
  • Table 13 Bacterial colonization at age 4 weeks: hazard-ratio estimates and significance Allele Ref. Heteroz. Rare Cod. Dom Rec. homoz. homoz. rs996165 C/T 1.00 2.07 3.89 0.00 0.01 0.01 rs3811215 A/C 1.00 0.84 0.43 0.45 0.38 0.26 rs227869 A/G 1.00 0.61 0.25 0.01 0.02 0.01 rsl0083416 A/G 1.00 0.72 0.27 0.10 0.11 0.07 rs227870 A/G 1.00 2.43 6.95 0.00 0.00 0.00 rsl483971 G/T 1.00 0.71 0.33 0.06 0.08 0.04 rs3751467 A/G 1.00 0.00 0.05 rs2128997 A/G 1.00 0.74 0.14 0.04 0.10 0.02 rs226985 C/T 1.00 2.08 4.27 0.00 0.00 0.00 rs2170810 A/G 1.00 2.15 4.62 0.00 0.00 0.00
  • Figure 14 shows multiple SNPs and their association with J45.
  • Figure 15 shows multiple SNPs and their association with exacerbation.
  • Figure 16 shows multiple SNPs and their association with exacerbation incidences.
  • Figure 17 shows multiple SNPs and their association with eczema.
  • Table 17 Time to eczema events: hazard-ratio estimates and significance
  • Figure 18 shows multiple SNPs and their association with asthma related events.
  • Table 18 Time to asthma related events: hazard-ratio estimates and significance
  • Table 19 summarizes the data above and shows the association of SNP markers in the vicinity of the TCRA gene on chr 14 with bacterial colonization of the newborn/infant airway.
  • Data presented show chr location, basepair location, allele involved, frequency in affected vs frequency in unaffected controls, chisquare association value, P value for all bacteria, odds ratios for all bacteria, P values and odds ratios for individual bacterias, including staphylococcus, pneumococcus, moraxella, hemophilus and hemophilus/moraxella combined. As shown the association is strongest to "all" bacteria, and to hemphilus, moraxella and hemophilus/moraxella combined.
  • Figure 19 shows a significant association detected with haplotypes formed by block 1 which includes the most highly associated SNP rs227870.
  • a single haplotype (the second most common in Europeans) is highly associated with the colonization phenotype.
  • Table 21 Association of the TCRA SNPs (that associate with bacterial colonization) with asthma at the age of 5, 6 and 7 years for age. P values and odds ratios are presented.
  • TCRa novel variants that predispose to bacterial colonization of the airways of newborns and infants and associate robustly with the development of asthma in later life provide new targets for the development of agents to prevent and assess risk for the development of asthma.

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Abstract

La présente invention concerne des compositions et des procédés pour la détection d’un risque augmenté de développement d’asthme.
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