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WO2003064591A2 - Acides nucleiques et proteines abca8 et leurs utilisations - Google Patents

Acides nucleiques et proteines abca8 et leurs utilisations Download PDF

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Publication number
WO2003064591A2
WO2003064591A2 PCT/US2002/033496 US0233496W WO03064591A2 WO 2003064591 A2 WO2003064591 A2 WO 2003064591A2 US 0233496 W US0233496 W US 0233496W WO 03064591 A2 WO03064591 A2 WO 03064591A2
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abca8
protein
seq
nucleic acid
ofthe
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WO2003064591A3 (fr
Inventor
Michael C. Dean
Lynn Schriml
Rando L. Allikmets
M. Patrice Denefle
Marie-Francoise Rosier-Montus
Isabelle Arnould-Reguigne
Catherine Prades
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Aventis Pharma SA
US Department of Health and Human Services
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Aventis Pharma SA
US Department of Health and Human Services
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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

Definitions

  • the present disclosure is related to extra- and intra-cellular transport, including the mechanisms controlling transport, diseases that arise from defects in such mechanisms, and methods of influencing (either inhibiting, enhancing or otherwise changing) transport.
  • the ABC (ATP-binding cassette transporter) gene superfamily encodes active transporter proteins and constitutes a family of proteins that have been extremely well conserved through evolution, from bacteria to humans (Ames and Lecar, FASEB J. , 1992, 6, 2660-2666).
  • the ABC proteins are involved in extra- and intracellular membrane transport of various substrates, for example ions, amino acids, peptides, sugars, vitamins, or steroid hormones.
  • 11 members have been described as associated with human disease, such as ABCA1, ABCA4 (ABCR) and ABCC7, (CFTR) which are thought to be involved in Tangier disease (Bodzioch et al., Nat.
  • P-glycoprotein was first identified over 20 years ago in chemotherapeutic drug- resistant tumor cells, and is now known to be a major cause of multidrug resistance in many cancers (Van Asperen e/ ⁇ /., ./. Pharmaceut. Sci. 86:881-884, 1997, 1997; Tsuji, Therap. Drug Monitor. 20:588-590, 1998). These implications reveal the importance ofthe functional role ofthe ABC gene family. The discovery of a new family of ABC gene members is expected to provide new insights into the physiopathology and treatment of human diseases.
  • the prototype ABC protein binds ATP and uses the energy from ATP hydrolysis to drive the transport of various molecules across cell membranes.
  • the functional protein contains two ATP- binding domains (nucleotide binding fold, NBF) and two transmembrane (TM) domains.
  • NBF nucleotide binding fold
  • TM transmembrane domains.
  • the genes are typically organized as full transporters containing two of each domain, or half transporters with only one of each domain. Most full transporters are arranged in a TM-NBF-TM-NBF fashion (Dean et al, Curr Opin Genet, 1995, 5, 79-785).
  • ABC ABC gene sub-families
  • ABCA genes and OABP represent the only two sub-families of ABC genes that do not have identifiable orthologs in the yeast genome (Decottignies and Goffeau, Nat Genet. Feb; 15(2): 137-45, 1997; Michaelis and Berkower, Cold Spring Harb Symp Quant Biol. 60:291-307, 1995). There is, however, at least one ⁇ £C4-related gene in C. elegans (ced-7) and several in Drosophila. Thus, the ABCA genes appear to have diverged after eukaryotes became multicellular and developed more sophisticated transport requirements.
  • ABCAl was demonstrated to be the gene responsible for Tangier disease, a disorder characterized by high levels of cholesterol in peripheral tissues, and a very low level of HDLs, and familial hypoalphalipoproteinemia (FHD) (Bodzioch et al. , Nat Genet ( 1999) 22, 347-51 ; Brooks- Wilson et al., Nat Genet (1999) 336-45; Rust et al., Nat Genet (1999) 22, 352-5; Marcil et al, The Lancet (1999) 354, 1341-46).
  • FHD familial hypoalphalipoproteinemia
  • the ABCAl protein is proposed to function in the reverse transport of cholesterol from peripheral tissues via an interaction with the apolipoprotein 1 (ApoA-1) of HDL tissues (see Wang et al, JBC 275(42): 33053-33058, 2000).
  • the ABCA2 gene is highly expressed in the brain, and ABCA3 in the lung, but no function has been ascribed to these loci.
  • the ABCA4 gene is exclusively expressed in the rod photoreceptors ofthe retina and mutations thereof are responsible for several pathologies of human eyes, such as retinal degenerative disorders (Allikmets et al, Science (1997) 277, 1805-1807; Allikmets et al, Nat Genet (1997) 15, 236-246; Sun et al, J Biol Chem (1999) 8269-81 ; Weng et al, Cell (1999) 98, 13- 23; Cremers et al, Hum Mol Genet (1998) 7, 355-362; Martinez-Mir et al, Genomics (1997) 40, 142-146).
  • ABCA4 is believed to transport retinal and/or retinal-phospholipid complexes from the rod photoreceptor outer segment disks to the cytoplasm, facilitating phototransduction.
  • Characterization of new genes from the ABCA subfamily is likely to yield biologically important transporters, which may have translocase activity for membrane lipid transport or the transport of other substances and which may play a role in human pathologies.
  • ABCA8 This disclosure describes the discovery and characterization of a new gene belonging to the ABCA protein sub-family, which has been designated ABCA8.
  • This new gene appears to be closely related to other ABCA subfamily members such as ABCAl.
  • ABCA8 is proposed to be involved in the energy-dependent transport of one or a variety of substances, for example ions, metals, amino acids, lipids, peptides, sugars, vitamins and steroid hormones.
  • the newly discovered gene also shows considerable conservation ofthe amino acid sequences when compared to previously known ABCA genes, particularly within the transmembrane region (TM) and the ATP-binding regions (NBF), and has a similar gene organization to ABCAl.
  • ABCA8 is one of a group of several ABCA genes that is organized in a single large cluster on chromosome 17q24, in a head-to-tail fashion. Furthermore, ABCA8 is transcribed with a tissue- specific distribution, but presents a heterogeneous pattern of expression, suggesting a regional and possibly functional specialization ofthe corresponding proteins.
  • the present disclosure relates to novel nucleic acids encoding ABCA8, for instance the
  • ABCA8 gene (SEQ ID NO: 1) and the predicted amino acid sequence (SEQ ID NO: 2).
  • the protein is believed to be involved in the reverse transport of cholesterol, as well as in the membrane transport of other lipophilic molecules, for instance inflammation mediating substances such as prostaglandins and prostacyclins, and in any pathology whose candidate chromosomal region is situated on chromosome 17, more precisely on the 17q arm and still more precisely in the 17q24 locus.
  • the disclosure also relates to means for the detection of polymo ⁇ hisms in general, and mutations in particular, in the ABCA8 gene or corresponding protein produced by allelic forms ofthe ABCA8 gene.
  • protein specific binding agents such as antibodies, that bind specifically to at least one epitope of an ABCA8 variant protein preferentially compared to wildtype ABCA8, and methods of using such antibodies in diagnosis and screening. Kits are also provided for carrying out the methods described herein.
  • Figure 1 represents a map ofthe 17q24 region containing the ABCA5, 6, 8-10 genes.
  • a physical map ofthe portion of chromosome 17q24 is shown in Figure 1A containing the 5 ABCA genes.
  • Figure 1A the location ofthe boundaries of BAC clones hRPK.235_I_10 and hRPK.293_K_20 (GenBank accession #s AC005495, AC005922) is indicated. Gene orientation is indicated by the arrows, and the size and location ofthe corresponding transcripts is shown below the map.
  • Figure 1 B shows a prediction ofthe intron and exon sequence locations within ABCA5, 10, 6, 9, and 8 in genomic DNA.
  • Figure 2 represents the alignment of ABC 1 -like genes.
  • An alignment ofthe amino acid sequences ofthe full-length ABCA10 (huest698739), 8 (huest990006), 9 (huest640918), and 6 (huestl55051) open reading frames, the partial sequence of ABCA5 (huest90625) and a gene originally called ABCAl 1, that is now known to be a pseudogene (huestl 133530) are shown as aligned.
  • Figure 3 displays two phylogenetic linkage trees of ABC 1 -like sequences. Phylogenetic trees were constructed with the alignments ofthe N- and C- terminal ATP-binding domains' sequence by both Neighbor joining (Figure 3A) and maximum parsimony (Figure 3B) methods.
  • Figure 4 shows the alignment ofthe full length ABCA8 cDNA (Figure 4A) and protein (Figure 4B) with the putative and incomplete ABCA8 cDNA and protein previously published in GenBank (referred to in the Figure as "Ref”; Accession numbers NM_007168 and NP_009099.1, respectively).
  • nucleic and amino acid sequences listed in the accompanying sequence Listings are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 shows the nucleic acid of human ABCA8 cDNA and the corresponding deduced amino acid sequence.
  • SEQ ID NO: 2 shows the amino acid sequence ofthe human ABCA8 protein encoded by
  • SEQ ID NO: 3 and SEQ ID NO: 4 show the nucleic acid sequences of two primers that can be used to amplify SEQ ID NO: 1 using the polymerase chain reaction PCR.
  • SEQ ID NO: 5 and SEQ ID NO: 6 show the nucleic acid sequences of two primers that can be used to amplify the region of SEQ ID NO: 1 corresponding to the ABCA8 protein coding region.
  • SEQ ID NO: 7 and SEQ ID NO: 8 show the nucleic acid sequences of two primers that can be used to amplify a particular region within ABCA8.
  • SEQ ID NO: 9 and SEQ ID NO: 10 show the nucleic acid sequences of two primers that can be used to amplify an ABCA8 encoding sequence.
  • BAC bacterial artificial chromosome bp base pair(s)
  • DNA deoxyribonucleic acid
  • gDNA genomic DNA
  • pfu plaque forming unit
  • ELISA enzyme-linked immunosorbant assay
  • TAE Tris acetate EDTA
  • Altered expression Expression of a nucleic acid (e.g., mRNA or protein) in a subject or biological sample from a subject that deviates from expression in a subject or biological sample from a subject having normal characteristics for the biological condition associated with the nucleic acid.
  • Normal expression can be found in a control, a standard for a population, etc.
  • the altered expression manifests as a transporter disease condition, such as deficient extra- or intracellular transport
  • characteristics of normal expression might include an individual who is not suffering from the transport disorder, a population standard of individuals believed not to be suffering from the disease, etc.
  • certain altered expression such as altered ABCA8 nucleic acid or ABCA8 protein expression, can be described as being associated with the biological conditions of altered (e.g., reduced) transporter function and tendency to develop a transporter deficiency.
  • altered expression may be associated with a disease.
  • the term "associated with” includes an increased risk of developing the disease as well as the disease itself.
  • Altered protein expression such as altered ABCA8 protein expression, also refers to expression of a protein that is in some manner different to expression ofthe protein in a normal (wild type) situation. This includes but is not necessarily limited to: (1) a mutation in the protein such that one or more ofthe amino acid residues is different; (2) a short deletion or addition of one or a few amino acid residues to the sequence ofthe protein; (3) a longer deletion or addition of amino acid residues, such that an entire protein domain or sub-domain is removed or added; (4) expression of an increased amount ofthe protein, compared to a control or standard amount; (5) expression of an decreased amount ofthe protein, compared to a control or standard amount; (6) alteration ofthe subcellular localization or targeting ofthe protein; (7) alteration ofthe temporally regulated expression ofthe protein (such that the protein is expressed when it normally would not be, or alternatively is not expressed when it normally would be); and (8) alteration ofthe localized (e.g., organ or tissue specific) expression ofthe protein (such that the protein is
  • Amplification When used in reference to a nucleic acid, techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen.
  • An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization ofthe primers to nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.
  • the product of in vitro amplification can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques.
  • Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBATM RNA transcription-free amplification (see U.S. Patent No. 6,025,134).
  • Double-stranded DNA has two strands, a 5' -> 3' strand, referred to as the plus strand, and a 3' -> 5' strand (the reverse compliment), referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5' -> 3' direction, the minus strand ofthe DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand and identical to the plus strand (except that U is substituted for T).
  • Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA.
  • Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA.
  • Antigene molecules are either antisense or sense molecules directed to a dsDNA target.
  • Binding or stable binding An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount ofthe oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties ofthe target:oligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including both functional and physical binding assays. Binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation and the like.
  • Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures.
  • one widely method involves observing a change in light abso ⁇ tion of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in abso ⁇ tion at a characteristic temperature as the oligonucleotide (or analog) and the target disassociate from each other, or melt.
  • T m The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (T m ) at which 50% ofthe oligomer is melted from its target.
  • T m the temperature at which 50% ofthe oligomer is melted from its target.
  • a higher (T m ) means a stronger or more stable complex relative to a complex with a lower (T m ).
  • Biological condition Designates a condition of a subject that can be assessed through observation or through the analysis of a biological sample, e.g., altered expression level of ABCA8 protein in comparison to a control expression level, or ability of cells from a subject to transport cholesterol.
  • Biological sample Any sample in which the presence of a protein and/or ongoing expression of a protein may be detected.
  • Suitable biological samples include samples containing genomic DNA or RNA (including mRNA), obtained from body cells of a subject, such as but not limited to those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.
  • Cassette A segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion ofthe cassette in the proper reading frame for transcription and translation.
  • cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments
  • cDNA can also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule.
  • UTRs untranslated regions
  • Coding sequence A DNA sequence that is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences include DNA regulatory sequences, such as promoters, enhancers, terminators, and so forth, which provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a coding sequence is under the control of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • Corresponding to is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
  • a nucleic acid or amino acid sequence alignment may include spaces.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the repeating units in DNA polymers are four different nucleotides, each of which comprises one ofthe four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides referred to as codons
  • codon code for each amino acid in a polypeptide.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Except where single-strandedness is required by the text herein, DNA molecules, though written to depict only a single strand, encompass both strands of a double-stranded DNA molecule. Thus, a reference to the nucleic acid molecule that encodes a specific protein, or a fragment thereof, encompasses both the sense strand and its reverse complement. Thus, for instance, it is appropriate to generate probes or primers from the reverse complement sequence ofthe disclosed nucleic acid molecules.
  • Deletion The removal of a sequence of DNA, the regions on either side being joined together.
  • a polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
  • the anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • Functional fragments and variants of a polypeptide Included are those fragments and variants that maintain at least one function ofthe parent polypeptide. It is recognized that the gene or cDNA encoding a polypeptide can be considerably mutated without materially altering one or more the polypeptide's functions.
  • the genetic code is well known to be degenerate, and thus different codons encode the same amino acids.
  • the mutation can be conservative and have no material impact on the essential functions of a protein (see Stryer, Biochemistry 4 th Ed., W. Freeman & Co., New York, NY, 1995).
  • part of a polypeptide chain can be deleted without impairing or eliminating all of its functions, e.g., sequence variants a protein, such as a 5' or 3' variant, may retain the full function of an entire protein.
  • insertions or additions can be made in the polypeptide chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et al, Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1998).
  • Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications or the inco ⁇ oration of unusual amino acids.
  • Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquination, labeling, e.g., with radionucleides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art.
  • a variety of methods for labeling polypeptides and labels useful for such pu ⁇ oses are well known in the art, and include radioactive isotopes such as 32 P, ligands that bind to or are bound by labeled specific binding partners (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands.
  • Functional fragments and variants can be of varying length. For example, a fragment may consist of 10 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more amino acid residues.
  • a functional fragment or variant of ABCA8 is defined herein as a polypeptide that is capable of transporter activity, including any polypeptide six or more amino acid residues in length that is capable of transporter activity.
  • Gene refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA corresponding to a polypeptide.
  • Heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site ofthe cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as "base pairing.” More specifically, A will hydrogen bond to T or U, and G will bond to C.
  • base pairing More specifically, A will hydrogen bond to T or U, and G will bond to C.
  • “Complementary” refers to the base pairing that occurs between to distinct nucleic acid sequences or two distinct regions ofthe same nucleic acid sequence.
  • specifically hybridizable and specifically complementary are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target.
  • the oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable.
  • An oligonucleotide or analog is specifically hybridizable when binding ofthe oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide or analog to non-target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature ofthe hybridization method of choice and the composition and length ofthe hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na + concentration) ofthe hybridization buffer will determine the stringency of hybridization, though waste times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 1 1 , herein inco ⁇ orated by reference. The following is an exemplary set of hybridization conditions and is not meant to be limiting.
  • Hybridization 5x-6x SSC at 65°C-70°C for 16-20 hours
  • Hybridization 6x SSC at RT to 55°C for 16-20 hours
  • hybridization conditions described above are adapted to hybridization, under high stringency conditions, of a molecule of nucleic acid of varying length from 20 nucleotides to several hundreds of nucleotides.
  • the hybridization conditions described above may be adjusted as a function ofthe length ofthe nucleic acid whose hybridization is sought or ofthe type of labeling chosen, according to techniques known to one skilled in the art. Suitable hybridization conditions may, for example, be adjusted according to the teaching contained in the manual by Hames and Higgins, Gene Transcription: A practical approach Transcription and Translation, IRL/Oxford University Press, 1993.
  • Hypercholesterolemia or high cholesterol, is a high level of cholesterol in the blood, which can cause an arterial plaque to form and accumulate, leading to blockages in the arteries (atherosclerosis), increasing the risk for heart attack, stroke, circulation problems, and death.
  • Hypercholesterolemia is measured relative to normal ranges or cholesterol obtained from population based studies.
  • In vitro amplification includes techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen.
  • An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization ofthe primers to nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies ofthe nucleic acid.
  • the product of in vitro amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques.
  • in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,31 1); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBATM RNA transcription-free amplification (see U.S. Patent No. 6,025,134).
  • Isolated for the pu ⁇ oses ofthe present disclosure designates that a biological material (nucleic acid or protein) has been substantially separated from or purified away from other biological components in the cell or the organism in which the compound naturally occurs i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • a biological material nucleic acid or protein
  • a polynucleotide present in the natural state in a plant or an animal is not isolated.
  • the same nucleotide separated from the cell and adjacent nucleic acids in which it is naturally inserted in the genome ofthe plant or animal is considered “isolated.”
  • Labeled A biomolecule attached covalently or noncovalently to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various pu ⁇ oses are discussed, e.g., in Sambrook et al, Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989 and Ausubel et al, Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1998.
  • ATP can be labeled in any one of its three phosphate groups with radioisotopes such as 32 P or 33 P, or in its sugar moiety with radioisotopes such as 35 S.
  • Mammal This term includes both human and non-human mammals. Similarly, the term subject includes both human and veterinary subjects.
  • Modulator An agent that increases or decreases (modulates) the activity of a protein as measured by the change in an experimental parameter.
  • a modulator can be essentially any compound, such as a chemotherapeutic agent, a polypeptide, a hormone, a nucleic acid, a sugar, a lipid and the like.
  • Mutation Any change ofthe DNA sequence within a gene or chromosome. In some instances, a mutation will alter a characteristic or trait (phenotype), but this is not always the case. Types of mutations include base substitution point mutations (e.g., transitions or transversions), deletions, and insertions.
  • Missense mutations are those that introduce a different amino acid into the sequence ofthe encoded protein; nonsense mutations are those that introduce a new stop codon.
  • mutations can be in-frame (not changing the frame ofthe overall sequence) or frame shift mutations, which may result in the misreading of a large number of codons (and often leads to abnormal termination ofthe encoded product due to the presence of a stop codon in the alternative frame).
  • This term specifically encompasses variations that arise through somatic mutation, for instance those that are found only in disease cells, but not constitutionally, in a given individual.
  • somatically-acquired variations include the point mutations that frequently result in altered function of various genes that are involved in development of cancers.
  • This term also encompasses DNA alterations that are present constitutionally, that alter the function ofthe encoded protein in a readily demonstrable manner, and that can be inherited by the children of an affected individual.
  • the term overlaps with "polymo ⁇ hism," as defined below, but generally refers to the subset of constitutional alterations that have arisen within the past few generations in a kindred and that are not widely disseminated in a population group.
  • the term is directed to those constitutional alterations that have major impact on the health of affected individuals.
  • Nucleotide This term includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA).
  • a nucleotide is one monomer in a polynucleotide.
  • the term nucleotide designates both the natural nucleotides (A, T, G, C) as well as the modified nucleotides that comprise at least one modification such as (1) an analog of a purine, (2) an analog of a pyrimidine, or (3) an analogous sugar, examples of such modified nucleotides being described, for example, in the PCT application No.
  • a first polynucleotide is considered as being complementary to a second polynucleotide when each base ofthe first nucleotide is paired with the complementary base ofthe second polynucleotide whose orientation is reversed.
  • the complementary bases are A and T (or A and U), or C and G.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide or nucleic acid.
  • a nucleic acid is a polymeric compound comprised of covalently linked subunits called nucleotides.
  • Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded.
  • DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides that encodes a protein is called the sense sequence or coding sequence.
  • a nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary structure ofthe molecule, and does not limit it to any particular tertiary form.
  • this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a recombinant DNA molecule is a DNA molecule that has undergone a molecular biological manipulation
  • Oligonucleotide A plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length.
  • An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions.
  • oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.
  • Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • 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 encoding an ABCA8 polypeptide ofthe disclosure.
  • oligonucleotides (one or both of which may be labeled) can be used as in vitro amplification primers, either for cloning full lengths or fragments of ABCA8 nucleic acids, or to detect the presence of nucleic acids encoding ABCA8.
  • an oligonucleotide ofthe disclosure can form a triple helix with ABCA8 DNA molecules.
  • oligonucleotides are prepared synthetically, in some embodiments on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
  • Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases.
  • a sequence such as DNA or RNA
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression ofthe coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Open reading frame A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.
  • Ortholog Two nucleic acid or amino acid sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.
  • compositions and formulations suitable for pharmaceutical delivery of compounds and molecules herein disclosed are conventional. Martin, Remington 's Pharmaceutical Sciences, published by Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of compounds and molecules herein disclosed.
  • nucleic acid, polypeptide, vector, or host cell ofthe disclosure will in some embodiments be introduced in vivo in a pharmaceutically acceptable vehicle or excipient.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and so forth, when administered to a subject.
  • pharmaceutically acceptable means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • excipient refers to a diluent, adjuvant, or vehicle with which the compound is administered.
  • Such pharmaceutical excipients or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and so forth.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are in some embodiments employed as excipients or carriers, particularly for injectable solutions.
  • the disclosure contemplates delivery of a vector that will express a therapeutically effective amount of ABCA8 polypeptide for gene therapy applications.
  • therapeutically effective amount is used herein to mean an amount sufficient to reduce by at least about 15 percent, in some embodiments by at least 50 percent, in other embodiments by at least 75, 80, or 90 percent, and in still other embodiments entirely prevent, a clinically significant deficit in the activity, function and response ofthe host.
  • a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host.
  • Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds.
  • polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins.
  • polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
  • polypeptide fragment refers to a portion of a polypeptide that exhibits at least one useful epitope.
  • functional fragments of a polypeptide refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, smaller peptides containing the biological activity of insulin, or conservative variants ofthe insulin, are thus included as being of use.
  • soluble refers to a form of a polypeptide that is not inserted into a cell membrane.
  • Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T);
  • I Isoleucine
  • L Leucine
  • M Methionine
  • V Valine
  • F Phenylalanine
  • Y Tyrosine
  • W Tryptophan
  • an equivalent amino acid according to the present disclosure will be understood to mean for example replacement of a residue in the L form by a residue in the D form or the replacement of a glutamic acid (E) by a pyro-glutamic acid according to techniques well known to persons skilled in the art.
  • two amino acids belonging to the same class that is to say two uncharged polar, nonpolar, basic or acidic amino acids, are also considered as equivalent amino acids.
  • Variations in the cDNA sequence that result in amino acid changes, whether conservative or not, are usually minimized in order to preserve the functional and immunologic identity ofthe encoded protein.
  • the immunologic identity ofthe protein may be assessed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved.
  • Any cDNA sequence variant will preferably introduce no more than twenty, and preferably fewer than ten amino acid substitutions into the encoded polypeptide.
  • Variant amino acid sequences may, for example, be 80, 90 or even 95% or 98% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI website.
  • a variant polypeptide includes any analogue, fragment, derivative, or mutant that is derived from a polypeptide or protein and that retains at least one biological property ofthe polypeptide or protein.
  • Different variant polypeptides or variant proteins may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences ofthe structural gene coding for the protein, or may involve differential splicing or post-translational modification.
  • Variant polypeptides also include a related (homologous) protein having substantially the same biological activity, but that has been obtained from a different species.
  • a variant polypeptide may contain up to 3, up to 5, up to 10, up to 15, or up to 25 or more different, deleted or added amino acid residues.
  • variant polypeptides having single or multiple amino acid substitutions, deletions, additions, or replacements.
  • These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non- conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more ofthe amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin, and a combination of these changes.
  • the techniques for obtaining these variant polypeptides including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.
  • Polymorphism Variant in a sequence of a gene, usually carried from one generation to another in a population. Polymo ⁇ hisms can be those variations (nucleotide sequence differences) that, while having a different nucleotide sequence, produce functionally equivalent gene products, such as those variations generally found between individuals, different ethnic groups, geographic locations.
  • polymo ⁇ hism also encompasses variations that produce gene products with altered function, i.e., variants in the gene sequence that lead to gene products that are not functionally equivalent. This term also encompasses variations that produce no gene product, an inactive gene product, or increased or increased activity gene product.
  • Polymo ⁇ hisms can be referred to, for instance, by the nucleotide position at which the variation exists, by the change in amino acid sequence caused by the nucleotide variation, or by a change in some other characteristic ofthe nucleic acid molecule or protein that is linked to the variation (e.g., an alteration of a secondary structure such as a stem-loop, or an alteration ofthe binding affinity ofthe nucleic acid for associated molecules, such as polymerases, RNases, and so forth).
  • Probes and primers Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this disclosure.
  • a probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
  • Methods for labeling and guidance in the choice of labels appropriate for various pu ⁇ oses are discussed, e.g., in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992).
  • Primers are short nucleic acid molecules, preferably DNA oligonucleotides 10 nucleotides or more in length. More preferably, longer DNA oligonucleotides can be about 15, 17, 20, or 23 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • PCR polymerase chain reaction
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that pu ⁇ ose such as Primer (Version 0.5, ⁇ 1991 , Whitehead Institute for Biomedical Research, Cambridge, MA).
  • probes and primers can be selected that comprise at least 17, 20, 23, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of ABCA8 nucleotide sequences.
  • the disclosure thus includes isolated nucleic acid molecules that comprise specified lengths ofthe disclosed ABCA8 cDNA sequences.
  • Such molecules can comprise at least 17, 20, 23, 25, 30, 35, 40, 45 or 50 or more consecutive nucleotides of these sequences, and can be obtained from any region ofthe disclosed sequences.
  • the ABCA8 cDNA sequences can be apportioned into halves, thirds or quarters based on sequence length, and the isolated nucleic acid molecules can be derived from the first or second halves ofthe molecules, from any ofthe three thirds or any ofthe four quarters.
  • the human ABCA8 cDNA, ORF, coding sequence and gene sequences can be apportioned into about halves, thirds or quarters based on sequence length, and the isolated nucleic acid molecules (e.g., oligonucleotides) can be derived from the first or second halves ofthe molecules, from any ofthe three thirds, or any ofthe four quarters.
  • the cDNA also could be divided into smaller regions, e.g. about eighths, sixteenths, twentieths, fiftieths and so forth, with similar effect.
  • nucleic acid molecules for instance to be used as hybridization probe molecules, may be selected from the region encoding the N-terminal nucleotide binding domain (NBD1) region (e.g., about residues 502-656, or a fragment thereof), or from the region encoding the C-terminal nucleotide binding domain (NBD2) (e.g., about residues 1308-1621 or a fragment thereof), or the or the region encoding the N-terminal transmembrane domain (TM 1 ) region (e.g., about residues 1-501, or a fragment thereof) or the region encoding the C-terminal transmembrane domain (TM2) region (e.g., about residues 656-1307) ofthe amino acid sequence encoding the human ABCA8 cDNA shown in SEQ ID NO
  • Another mode of division is to select the 5' (upstream) and/or 3' (downstream) region associated with an ABCA8 gene, or to select an intron or portion thereof.
  • Promoter A binding site in a DNA chain at which RNA polymerase binds to initiate transcription of messenger RNA by one or more nearby structural genes.
  • a promoter contains the necessary elements for the transcription and translation of a protein-coding sequence.
  • An expression vector containing the necessary elements for the transcription and translation of a protein-coding sequence may function as a promoter.
  • nucleotide sequence coding for the ABCA8 polypeptide or antigenic fragment, derivatives or analogs thereof, or functionally active derivatives, including chimeric proteins thereof may be inserted into an expression vector containing the elements of a promoter, including a "promoter sequence," i.e., a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • promoter sequence i.e., a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • nucleic acids encoding the ABCA8 polypeptide ofthe disclosure can be operationally associated with a promoter in an expression vector ofthe disclosure. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also includes a replication origin.
  • purified In a more pure form than is found in nature.
  • the term purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell.
  • substantially purified as used herein refers to a molecule (e.g., a nucleic acid, polypeptide, oligonucleotide, etc.) that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the molecule is a polypeptide that is at least 50% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • polypeptide is at least at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet other embodiments, the polypeptide is at least 90% or at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • Recombinant A nucleic acid that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • regulatory region means a nucleic acid sequence that regulates the expression of a nucleic acid.
  • a regulatory region may include sequences that are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin (a heterologous or recombinant region, responsible for expressing different proteins or even synthetic proteins).
  • the regulatory sequences can be sequences of eukaryotic or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific manner, or in an inducible or non-inducible manner.
  • Regulatory regions include origins of replication, RNA splice sites, enhancers, transcriptional termination sequences, signal sequences that direct the polypeptide into the secretory pathways ofthe target cell, and promoters.
  • a regulatory region from a heterologous source is a regulatory region that is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences that do not occur in nature, but that are designed for instance by an ordinarily skilled artisan.
  • a selection marker or detectable marker is a biochemical characteristic or function of a molecule that allows it to be identified based upon its performance, e.g., ⁇ - galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.).
  • a recombinant vector/host system can be identified and selected based upon the presence or absence of certain selection marker functions, caused by the insertion of foreign genes in the vector.
  • recombinants containing ABCA8 nucleic acid inserts can be identified by the absence of marker function (e.g., loss of antibiotic resistance).
  • Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms ofthe similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • homologs or orthologs ofthe ABCA8 protein, and the corresponding cDNA sequence will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or cDNAs are derived from species that are more closely related (e.g., human and chimpanzee sequences), compared to species more distantly related (e.g., human and C. elegans sequences).
  • ABCA8 orthologs are at least 90% identical at the nucleotide level and at least 98%) identical at the amino acid level when comparing ABCA8 to an orthologous ABCA8.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215: 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
  • NCBI National Center for Biotechnology Information
  • the Search Tool can be accessed at the NCBI website, together with a description of how to determine sequence identity using this program.
  • Nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy ofthe genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
  • Specific binding agent An agent that binds substantially only to a defined target.
  • ABCA8 protein-specific binding agent binds substantially only an ABCA8 protein.
  • the phrase ABCA8 protein-specific binding agent includes anti-ABCA8 protein antibodies and other agents (such as soluble receptors) that bind substantially only to an ABCA8 protein, such as the ABCA8 proteins ofthe disclosure, or conservative variants thereof.
  • Anti-ABCA8 protein antibodies can be produced using standard procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent binds substantially only to the ABCA8 protein can readily be made by using or adapting routine procedures.
  • a phosphospecific binding agent specifically binds to a peptide containing a phosphorylated residue.
  • Shorter fragments of antibodies can also serve as specific binding agents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to ABCA8 would be ABCA8-specific binding agents.
  • antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment ofthe antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region ofthe light chain and the variable region ofthe heavy chain expressed as two chains; and (6) single chain antibody (SCA), a genetically engineered molecule containing the variable region ofthe light chain, the variable region ofthe heavy chain, linked by a suitable polypeptide linker as a genetically
  • Target sequence is a portion of ssDNA, dsDNA or RNA that, upon hybridization to a therapeutically effective oligonucleotide or oligonucleotide analog, results in the inhibition of expression. For example, hybridization of therapeutically effectively oligonucleotide to an ABCA8 target sequence results in inhibition of ABCA8 expression.
  • an antisense or a sense molecule can be used to target a portion of dsDNA, as both will interfere with the expression of that portion ofthe dsDNA.
  • the antisense molecule can bind to the plus strand, and the sense molecule can bind to the minus strand.
  • target sequences can be ssDNA, dsDNA, and RNA.
  • Transfected A process by which a nucleic acid molecule is introduced into a cell, for instance by molecular biology techniques, resulting in a transfected cell.
  • transfection encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transfection with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.
  • a cell has been transformed by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change.
  • the transforming DNA may be integrated (covalently linked) into chromosomal DNA making up the genome ofthe cell.
  • Treating a disease Includes inhibiting or preventing the partial or full development or progression of a disease, for example in a person who is known to have a predisposition to a disease.
  • An example of a person with a known predisposition is someone with a history of diabetes in his or her family, or who has been exposed to factors that predispose the subject to a condition, such as lupus or rheumatoid arthritis.
  • treating a disease refers to a therapeutic intervention that ameliorates at least one sign or symptom of a disease or pathological condition, or interferes with a pathophysiological process, after the disease or pathological condition has begun to develop.
  • Variant nucleic acids are understood to mean a nucleic acid that differs by one or more bases relative to the reference polynucleotide.
  • a variant nucleic acid may be of natural origin, such as an allelic variant that exists naturally, or it may be a non-natural variant obtained, for example, by mutagenic techniques.
  • the differences between the reference (for instance, wild-type) nucleic acid and the variant nucleic acid are small such that the nucleotide sequences ofthe reference nucleic acid and ofthe variant nucleic acid are very similar and, in many regions, identical.
  • the nucleotide modifications present in a variant nucleic acid may be silent, which means that they do not alter the amino acid sequences encoded by the variant nucleic acid.
  • nucleic acid variants will contain up to 5 different residues compared to the reference sequence, in other embodiments up to 10, up to 25, up to 50 or more.
  • nucleotides in a variant nucleic acid may also result in substitutions, additions or deletions in the polypeptide encoded by the variant nucleic acid in relation to the polypeptides encoded by the reference nucleic acid.
  • nucleotide modifications in the coding regions may produce conservative or non-conservative substitutions in the amino acid sequence ofthe polypeptide.
  • Such variant polypeptides in some embodiments will contain no more than 3 amino acid changes, no more than 5, no more than 10, no more than 15, or in some instances 25 or more differences than a reference sequence.
  • the variant nucleic acids according to the disclosure encode polypeptides that substantially conserve the same function or biological activity as the polypeptide of the reference nucleic acid or alternatively the capacity to be recognized by antibodies directed against the polypeptides encoded by the initial reference nucleic acid.
  • variant nucleic acids will thus encode mutated forms ofthe polypeptides whose systematic study will make it possible to deduce structure-activity relationships ofthe protein in question. Knowledge of these variants in relation to the disease studied is important as it helps to elucidate the molecular cause ofthe pathology.
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell.
  • Recombinant DNA vectors are vectors having recombinant DNA.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements known in the art.
  • Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.
  • a polynucleotide may be included in a vector and/or such a polynucleotide may be included in a composition and remains nevertheless in the isolated state because ofthe fact that the vector or the composition does not constitute the polynucleotide's natural environment.
  • Specific embodiments include purified proteins having high identity (e.g., 98%) to SEQ ID NO: 2.
  • the disclosure includes oligonucleotides having high identity (e.g., 80%, 90%) to residues 597-636 of
  • ABCA8 proteins have ABCA8 protein biological activity, for instance in that they can complement an ABCA8 null phenotype.
  • Specific embodiments include ABCA8 proteins having biological activity as extra- and/or intracellular membrane transport proteins that can complement ABCA8 null phenotypes by ameliorating the respective transport deficiencies.
  • Specific binding agents that bind to epitopes ofthe ABCA8 protein and to specific regions within the protein (e.g., residues 597-636 of SEQ ID NO: 2) are provided. Methods to diagnose and detect defects or alterations in ABCA8 expression are provided, as are methods for screening for specific binding agents of ABCA8.
  • Transformed cells, recombinant vectors and transgenic animals containing the disclosed sequences are provided.
  • the methods are used to detect hypercholesterolemia, drug resistance, retinal degeneration, defective infra- or extracellular transport of ions, amino acids, peptides, sugars, vitamins or steroid hormones, or neurological disease.
  • Other methods are used to detect chemotherapy resistant cells, or employ primers having sequences identical to at least 10 contiguous nucleotides of disclosed sequences, including SEQ ID NOS: 1, 81, 20, 33, 48, 61 or 77.
  • Antibodies specific to ABCA8, and the use of such antibodies are disclosed.
  • Kits for using the ABCA8 protein are disclosed, including kits for screening defects in ABCA8 biological activity, and kits used to assay particular defects in individuals with defective ABCA8, including altered extra- or intracellular transport.
  • Components useful in the provided kits are disclosed, including nucleotide probes, nucleic acid molecules, reagents of use, and instructions for using the kits.
  • compositions including polypeptides encoding the ABCA8 protein are provided.
  • ABCA8 a novel human ABCA-family gene
  • the new gene is a member of an ABCA gene cluster that is closely spaced and arranged head-to-tail at a single region of chromosome 17q24, all five members of this cluster encode putative full transporters (see Figure 1).
  • the ABCA8 gene encodes a protein proposed to be involved in the energy-dependent transport of one or a variety of substances, for example ions, metals, amino acids, lipids, peptides, sugars, vitamins and steroid hormones.
  • the newly discovered gene also shows considerable conservation ofthe amino acid sequences compared to other known ABCA genes, particularly within the transmembrane region (TM) and the ATP-binding regions (NBF), and has a similar gene organization to ABCAl.
  • the full-length cDNA for human ABCA8 (SEQ ID NO: 1) is 5797 base pairs long, which is somewhat longer than the ABCA8 sequence previously released in the GenBank database (Accession # NM 007168, corresponding to putative protein with Accession # NP 09099.1).
  • the ABCA8 ORF encodes a protein of 1621 amino acids, having a predicted molecular weight of approximately 184 kDa and a predicted pi of about 7.11.
  • the human ABCA8 cDNA as identified herein includes a region of 120 residues, corresponding to nucleotides 1927-2046 of SEQ ID NO: 1 and encoding the amino acids at residues 597-636 of SEQ ID NO: 2, that were previously completely unknown. This newly identified region overlaps the first Nucleotide Binding Fold (NBFl) of ABCA8, and it is believed that the protein previously available in GenBank (Accession # NP_009099.1) is non-functional or dis-functional for ABC transporter activity due to the lack ofthe complete NBFl .
  • NBFl Nucleotide Binding Fold
  • the human ABCA8 protein comprises the deduced protein sequence (SEQ ID NO: 2) corresponding to the herein described human ABCA8 cDNA sequence (SEQ ID NO: 1). This protein sequence shows high sequence homology to other members ofthe ABCA family.
  • Figure 2 shows an alignment ofthe provided ABCA8 sequence (referred to in the figure as huest990006) and other members ofthe ABCA gene family.
  • the ABCA8 protein contains the following domains: (1) an N-terminal transmembrane domain (TM1) that falls within residues 1-501; (2) a Nucleotide Binding Domain (NBFl) that falls within residues 502-656; (3) a C-terminal transmembrane domain (TM2) that falls within residues 656-1307; (2) a second Nucleotide Binding Domain (NBF2) that falls within residues 1308-1621.
  • TM1 N-terminal transmembrane domain
  • NVFl Nucleotide Binding Domain
  • ABCA8 is a single-copy gene that is highly expressed in ovarian tissue.
  • the homology between the protein functional domains (e.g. transmembrane and nucleotide binding regions) of ABCA8 to other ABCA family members indicate that the protein product of ABCA8 is likely to be important in extra- and intracellular transport, as has been shown with ABCAl.
  • ABCA8 maps to mouse chromosome 11, 64-67 cM from the centromere.
  • ABCA8 cDNA sequences were used to pinpoint the location of ABCA8 in the human genome.
  • ABCA8 was localized by amplification of monochrome hybrids and radiation hybrids using the Polymerase Chain Reaction (PCR) (see Morten et al, Hum. Genet. 88(2): 200-203, 1991). Subsequently, the position of ABCA8 on the draft human genome map was determined using chromosomal assignment and somatic cell hybrids (CASH) (see Ryu et al., Mol Cells 10(5): 598-600, 2000).
  • RT-PCR analysis shows that ABCA8 is expressed in heart, ovary, testes and liver.
  • Tsuruoka et al found that Northern Blot analysis revealed that expression is found in most organs, and in the highest levels in hearth, skeletal muscle, and liver (see Tsuruoka et al, Biochem. Biophys Res. Commun. 298(1 ):41, 2002).
  • ABCA8 maps to mouse chromosome 11, 64-67 cM from the centromere. ABCA8 is therefore a positional candidate for pathologies linked to this chromosome.
  • ABCA8 transporter proteins and variants thereof and nucleic acid molecules encoding these proteins, including cDNA sequences.
  • these sequences are used for ameliorating, treating, detecting, prognosing, and diagnosing diseases and conditions believed to be associated with altered ABCA8 expression (based on homology to other ABCA family members), such as hypercholesterolemia, drug resistance, retinal degeneration, resistance to chemotherapy, or neurological disease.
  • the prototypical ABCA8 sequences e.g. SEQ ID NOS: 1 and 2 are human, and the use of these sequences to produce transgenic animals having increased or decreased levels of ABCA8 protein is provided, as are diagnostic methods to detect defects or alterations in ABCA8 expression or ABCA8 protein production.
  • the human ABCA8 cDNA as identified herein includes a region of 120 residues, corresponding to nucleotides 1927-2046 of SEQ ID NO: 1 and encoding the amino acids at residues 597-636 of SEQ ID NO: 2, that were previously completely unknown. This newly identified region overlaps the first Nucleotide Binding Fold (NBFl) of ABC A8, and it is believed that the protein previously available in GenBank (Accession # NP 009099.1) is non-functional or dis-functional for ABC transporter activity due to the lack ofthe complete NBFl .
  • NBFl Nucleotide Binding Fold
  • provided ABCA8 proteins have ABCA8 protein biological activity, for instance in that they can complement an ABCA8 null phenotype by ameliorating the associated transport deficiencies.
  • One specific embodiment is thus an isolated human ABCA8 protein predicted to have an estimated molecular weight of about 184 kDa, wherein the human ABCA8 protein comprises the amino acid sequence as set forth in SEQ ID NO: 2 (or a conservative variant thereof), the protein is associated with extra- and/or intracellular membrane transport, and it can complement an ABCA8 null phenotype by ameliorating the transport deficienc(ies).
  • an ABCA8 nucleic acid in another embodiment, has altered expression (e.g., increased or decreased expression, such as altered transcription of ABCA8 mRNA, a mutated or deleted expression product, improper subcellular localization of a protein, etc.) as compared to a control nucleic acid (e.g., a nucleic acid amplified, using positive control sequences, from a subject not suffering from the biological condition).
  • expression of an ABCA8 nucleic acid is more than 50%, more than 75%, more than 100%, more than 200%, or more than 300% elevated or decreased when compared to a suitable control.
  • Suitable controls include the expression level of ABCA8 in a subject having normal transporter activity in a known sample, or a standard value as assigned by one of ordinary skill in the art as a suitable standard value. It will be apparent to one of ordinary skill in the art that the cDNAs disclosed herein and sequences derived from these may be utilized in many applications, including but not limited to, studies ofthe expression ofthe ABCA8 gene and mutants thereof, studies ofthe function ofthe ABCA8 protein, the generation of antibodies to the ABCA8 protein, and diagnosis and therapy of ABCA8 deleted or mutated in subjects to prevent or treat the defects in cell and tissue development, such as transporter deficiency. Descriptions of applications using of ABCA8 cDNA, or fragments thereof, are therefore intended to comprehend the use ofthe ABCA8 nucleic acids and variants thereof.
  • ABCA8 mRNA has a unique expression pattern compared to other ABCs, suggesting that the corresponding protein may perform tissue-specialized functions. In effect, expression patterns showed that the ABCA8 transcript is almost ubiquitous, with the strongest expression found in ovarian tissues.
  • Table 1 Expression patterns of ABCA8 mRNA.
  • the cDNA sequence ABCA8 (SEQ ID NO: 1) consists of 5797 nucleotides and contains a
  • the cDNA molecule of human ABCA8 has the nucleotide sequence as set forth in SEQ ID NO: 2).
  • ID NO: 1 includes an open reading frame beginning from the nucleotide at position 139 (base A of the ATG codon for initiation of translation) to the nucleotide at position 5004 (base A ofthe TGA stop codon).
  • the ABCA8 cDNA composing SEQ ID NO: 1 encodes a full length ABCA8 polypeptide of 1621 amino acids corresponding to the amino acid sequence of SEQ ID NO: 2.
  • the ABCA8 protein also demonstrates high conservation with other members ofthe ABCA family, as set forth in Table 2 ( Figure 2), as well as in particular conserved regions within these proteins (Tables 3-6).
  • NBF2 Nucleotide Binding Domain 2
  • RNA is extracted from human cells by any one of a variety of methods well known to those of ordinary skill in the art (Sambrook et al, in Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989; and Ausubel et al, in Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992 provide descriptions of methods for RNA isolation).
  • primary cells are obtained from normal tissues.
  • cells are obtained from tissues from subjects exhibiting the effects of ABCA8 transporter deficiency.
  • cell lines derived from normal or transporter-deficient tissues are used as a source of such RNA.
  • the extracted RNA is then used, for example, as a template for performing reverse transcription (RT)-PCR amplification of cDNA.
  • RT-PCR reverse transcription
  • amplification primers will be made according to the portion(s) ofthe cDNA that is to be amplified.
  • primers may be chosen to amplify a segment of a cDNA or, in another embodiment, the entire cDNA molecule. Variations in amplification conditions may be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed for instance in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990).
  • the ABCA8 protein coding portion may be amplified using primers, such as SEQ ID NO: 5 and 6.
  • the primers set forth as SEQ ID NO: 3 and 4 can be used to amplify SEQ ID NO:
  • the primers set forth as SEQ ID NO: 7 and 8 can be used to amplify a particular region (see Example 3) within ABCA8; and the primers set forth as SEQ ID NO: 9 and 10 can be used to amplify an ABCA8 encoding sequence.
  • SEQ ID NO: 9 and 10 can be used to amplify an ABCA8 encoding sequence.
  • Orthologs of human ABCA8 can be cloned in a similar manner, where the starting material consists of cells taken from a non-human species. Orthologs will generally share at least 50% sequence homology with the disclosed human ABCA8 cDNA. Where the non-human species is more closely related to humans, the sequence homology will in general be greater.
  • Closely related orthologous ABCA8 molecules may share at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% sequence homology with the disclosed human sequences.
  • Oligonucleotides derived from the human ABCA8 cDNA sequence are encompassed within the scope ofthe present invention.
  • Such oligonucleotide primers may comprise a sequence of at least 10 consecutive nucleotides ofthe ABCA8 nucleic acid sequence.
  • oligonucleotide primers comprising at least 15, 25, 30, 35, 40, 45 or 50 or more consecutive nucleotides of these sequences may also be used. These primers for instance may be obtained from any region ofthe disclosed sequences.
  • the human ABCA8 cDNA, ORF and gene sequences may be apportioned into about halves or quarters based on sequence length, and the isolated nucleic acid molecules (e.g., oligonucleotides) may be derived from the first or second halves ofthe molecules, or any ofthe four quarters.
  • the human ABCA8 cDNA, shown in SEQ ID NO: 1, can be used to illustrate this example.
  • the human ABCA8 cDNA shown in SEQ ID NO: 1 is 5797 nucleotides in length and may be hypothetically divided into about halves (nucleotides 1-2899 and 2900-5797) or about quarters (nucleotides 1-1449, 1450-2899, 2900-4348 and 4349- 5797).
  • Another advantageous portion ofthe reported ABCA8 locus is about residue 2026 to about residue 3113 of SEQ ID NO: 1, or the reverse complement of this sequence.
  • Nucleic acid molecules may be selected that comprise at least 10, 15, 20, 25, 30, 35, 40, 50 or 100 or more consecutive nucleotides of any of these or other portions ofthe human ABCA8 cDNA, or ofthe 5' or 3' flanking regions.
  • variant ABCA8 proteins include proteins that differ in amino acid sequence from the human ABCA8 sequences disclosed but that share at least 65% amino acid sequence identity with the provided human ABCA8 protein. In other embodiments, other variants will share at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity.
  • the ABCA8 gene may be isolated by routine procedures, such as those provided in Example 1. For instance, the ABCA8 gene may be isolated by homology screening using the cDNA sequence and the BLAST program.
  • Direct sequencing using the "long-distance sequence method," of one or more BAC or PAC clones that contain the ABCA8 sequence can be employed.
  • the simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties. These so-called conservative substitutions are likely to have minimal impact on the activity ofthe resultant protein. In another embodiment, more substantial changes in transporter function or other protein features may be obtained by selecting amino acid substitutions that are less conservative than conservative substitutions.
  • such changes include changing residues that differ more significantly in their effect on maintaining polypeptide backbone structure (e.g., sheet or helical conformation) near the substitution, charge or hydrophobicity ofthe molecule at the target site, or bulk of a specific side chain.
  • polypeptide backbone structure e.g., sheet or helical conformation
  • a hydrophilic residue e.g., seryl or threonyl
  • a hydrophobic residue e.g., leucyl, isoleucyl, phenylalanyl, or valyl
  • a cysteine or proline is substituted for (or by) any other residue
  • a residue having an electropositive side chain e.g., lysyl, arginyl, or histadyl
  • an electronegative residue e.g., glutamyl or aspartyl
  • a residue having a bulky side chain e.g., phenylalanine
  • one lacking a side chain e.g., glycine
  • changes in transporter activity or other protein features may be obtained by mutating, substituting or deleting regions of ABCA8 that have a known function, or regions where the function is yet to be determined.
  • a NBD (nucleotide binding domain) motif of ABCA8 (corresponding to about residues 502-656 or 1308-1621 of SEQ ID NO: 2) can be deleted, substituted with the NBD of another protein or a synthetic NBD, or residues within one or both ofthe NBD motif can be mutated.
  • residues within the two ABCA8 transmembrane domains are mutated or deleted, or the TM is substituted with a transmembrane domain of another protein or a synthetic TM.
  • a mutation is made within residues 597-636 of SEQ ID NO: 2.
  • Variant ABCA8 encoding sequences may be produced by standard DNA mutagenesis techniques.
  • Ml 3 primer mutagenesis is performed.
  • DNA molecules and nucleotide sequences that are derivatives of those specifically disclosed herein, and which differ from those disclosed by the deletion, addition, or substitution of nucleotides while still encoding a protein that has at least 90% sequence identity with the human ABCA8 encoding sequence disclosed (SEQ ID NO: 1), are comprehended by this disclosure.
  • At least one or more, at least 5 or more, at least 10 or more, at least 15 or more, at least 20 or more, or at least 25 or more nucleotides are deleted, added, or substituted while still encoding a protein that has at least 90% sequence identity with the human ABCA8 encoding sequence disclosed (SEQ ID NO: 1).
  • more closely related nucleic acid molecules that share at least 95%, or at least 98%o nucleotide sequence identity with the disclosed ABCA8 sequences e.g., SEQ ID NO: 1 and 2
  • nucleic acid molecule sharing at least 80% or more sequence identity with residues 597-636 of SEQ ID NO: 2 are comprehended by this disclosure.
  • such variants may differ from the disclosed sequences by alteration ofthe coding region to fit the codon usage bias ofthe particular organism into which the molecule is to be introduced.
  • the coding region may be altered by taking advantage ofthe degeneracy ofthe genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence substantially similar to the disclosed human ABCA8 protein sequences.
  • the genetic code because ofthe degeneracy of the genetic code, four nucleotide codon triplets - (GCT, GCG, GCC and GCA) - code for alanine.
  • the coding sequence of any specific alanine residue within the human ABCA8 protein therefore, could be changed to any of these alternative codons without affecting the amino acid composition or characteristics ofthe encoded protein.
  • variant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences.
  • this disclosure also encompasses nucleic acid sequences that encode an ABCA8 protein, but which vary from the disclosed nucleic acid sequences by virtue ofthe degeneracy ofthe genetic code.
  • variants of ABCA8 may also be defined in terms of their sequence identity with the prototype human ABCA8 protein.
  • human ABCA8 proteins share at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with the human ABCA8 protein (SEQ ID NO: 2).
  • At least one or more, at least 5 or more, at least 10 or more, at least 15 or more, at least 20 or more, or at least 25 or more amino acids are deleted, added, or substituted while still encoding a protein that has at least 90% sequence identity with the ABCA8 encoding sequence (SEQ ID NO: 2).
  • Nucleic acid sequences that encode such proteins/fragments readily may be determined simply by applying the genetic code to the amino acid sequence of an ABCA8 protein or fragment, and such nucleic acid molecules may readily be produced by assembling oligonucleotides corresponding to portions ofthe sequence.
  • Nucleic acid molecules that are derived from the human ABCA8 cDNA nucleic acid sequences include molecules that hybridize under low stringency, high stringency, or very high stringency conditions to the disclosed prototypical ABCA8 nucleic acid molecules, and fragments thereof.
  • Human ABCA8 nucleic acid encoding molecules e.g., the cDNA shown in SEQ ID NO: 1, and nucleic acids comprising this sequence
  • orthologs and homologs of this sequence may be incorporated into transformation or expression vectors.
  • homologs ofthe ABCA8 gene may now be cloned from other species, such as the rat or a monkey, by standard cloning methods. Such homologs will be useful in the production of animal models demonstrating the formation and progression of a variety of tumors.
  • orthologous ABCA8 molecules will share at least 65% sequence identity with the human ABCA8 nucleic acid disclosed herein; and in other embodiments, more closely related orthologous sequences will share at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with this sequence.
  • orthologous ABCA8 molecules will share at least 65% sequence, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with residues 597-636 of SEQ ID NO: 2.
  • orthologous ABCA8 molecules share high (e.g., at least 80% or more) homology with residues 597-636 of SEQ ID NO: 2 and at least 90% homology with the remainder of SEQ ID NO: 2.
  • the DNA sequence ofthe full-length ABCA8 cDNA (SEQ ID NO: 1) can be manipulated in studies to understand the expression ofthe gene and the function of its product.
  • partial or full-length cDNA sequences, which encode ABCA8 protein may be ligated into bacterial expression vectors.
  • E. coli Escherichia coli
  • fusion proteins consisting of amino terminal peptides encoded by a portion ofthe E. coli lacZ or trpE gene linked to ABCA8 proteins may be used to prepare polyclonal and monoclonal antibodies against these proteins. Thereafter, these antibodies may be used in other embodiments to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein and to localize proteins in tissues and individual cells by immunofluorescence.
  • Such antibodies may be specific for epitope tags, which can be added to the expression construct for identification and/or purification pu ⁇ oses.
  • Intact native protein also may be produced in E. coli or other cell culture systems in large amounts for functional studies. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989). Such fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response.
  • native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome binding site upstream ofthe cloned gene. If low levels of protein are produced, additional steps may be taken to increase protein production; if high levels of protein are produced, purification is relatively easy.
  • Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors (Ruther and Muller-Hill, EMBOJ. 2:1791-1794, 1983), pEXl-3 (Stanley and Luzio, EMBO J. 3(6):1429-1434, 1984) and pMRlOO (Gray et al, Proc. Natl. Acad. Sci. USA 79(21):6598-6602, 1982).
  • Vectors suitable for the production of intact native proteins include pKC30 (Belfort et al, J. Biol. Chem.
  • ABCA8 fusion proteins may be isolated from protein gels, lyophilized, ground into a powder and used as an antigen.
  • the DNA sequence can also be transferred from its existing context to other cloning vehicles, such as other plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al, Science 236:806-812, 1987).
  • vectors may then be introduced into a variety of hosts including, but not limited to, somatic cells, and simple or complex organisms, such as, but not limited to, bacteria, fungi (Timberlake and Marshall, Science 244:1313-1317, 1989), invertebrates, plants, and animals (Pursel et al, Science 244:1281-1288, 1989), which cells or organisms are rendered transgenic by the introduction ofthe heterologous ABCA8 cDNA.
  • the cDNA sequence is ligated to heterologous promoters. In one specific, non-limiting embodiment it may be ligated to the simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg, Proc. Natl.
  • the stable integration ofthe chimeric gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, J. Mol. Appl. Genet. 1 :327-341, 1982) and mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).
  • biochemical selection such as neomycin (Southern and Berg, J. Mol. Appl. Genet. 1 :327-341, 1982) and mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).
  • cell lines expressing native ABCA8 are created.
  • cell lines expressing a mutant ABCA8 are created.
  • DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence- alteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with nucleic acid amplification. These techniques are known to those of ordinary skill in the art.
  • the ABCA8 cDNA sequence (or portions derived from it, such as a nucleic acid encoding a polypeptide including an amino acid sequence selected from SEQ ID NO: 2 or residues 597-636 of SEQ ID NO: 2) or a mini gene (a cDNA with an intron and its own promoter) may be introduced into eukaryotic expression vectors by conventional techniques. These vectors are designed to permit the transcription ofthe cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription ofthe cDNA and ensure its proper splicing and polyadenylation.
  • Vectors containing the promoter and enhancer regions ofthe SV40 or long terminal repeat (LTR) ofthe Rous Sarcoma virus and polyadenylation and splicing signal from SV40 are readily available (Mulligan et al, Proc. Natl. Acad. Sci. USA 78:1078-2076, 1981 ; Gorman et al, Proc. Natl. Acad. Sci USA 78:6777-6781, 1982).
  • the level of expression ofthe cDNA can be manipulated with this type of vector by using promoters that have different activities (for example, the baculovirus pAC373 can express cDNAs at high levels in 5.
  • frugiperda cells (Summers and Smith, in Genetically Altered Viruses and the Environment, Fields et al. (Eds.) 22:319-328, CSHL Press, Cold Spring Harbor, New York, 1985) or by using vectors that contain promoters amenable to modulation, for example, a recombinant adenoviral vector containing a nuclear lacZ gene driven by a human ventricular/slow muscle myosin light chain 1 promoter (Shi et al, Hum Gene Ther. 8(4):403-410, 1997).
  • the expression ofthe cDNA can be monitored in the recipient cells 24 to 72 hours after induction (transient expression).
  • Some vectors contain selectable markers such as the gpt (Mulligan and Berg, Proc. Natl.
  • the vectors can be maintained in the cells as episomal, freely replicating entities by using regulatory elements of viruses, such as papilloma (Sarver et al, Mol. Cell Biol. 1 :486-496, 1981) or Epstein-Barr (Sugden et al, Mol. Cell Biol. 5:410-413, 1985).
  • viruses such as papilloma (Sarver et al, Mol. Cell Biol. 1 :486-496, 1981) or Epstein-Barr (Sugden et al, Mol. Cell Biol. 5:410-413, 1985).
  • the vectors may contain an internal ribosomal entry site (IRES) between the cDNA and a marker gene, such as neomycin or enhanced green fluorescent protein (EGFP).
  • IRES allows for the simultaneous expression ofthe two elements from a single isoform. Ribosomes bind the isoform at both the 5' end to translate the cDNA and at the IRES to translate, in one specific, non-limiting embodiment, the antibiotic resistance marker, or in another specific, non-limiting embodiment, the fluorescent marker.
  • the bicistronic expression via the IRES sequence provides a high degree of correlation between the antibiotic resistance and stable expression ofthe cDNA. Alternatively, only cells expressing the cDNA will show green fluorescence. Thus, the use of expression vectors containing an IRES is an efficient way to select for cells expressing the cDNA of interest.
  • Recombinant expression vectors can be introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, Virology 52(2):456-467, 1973) or strontium phosphate (Brash et al, Mol. Cell Biol. 7(5):2031- 2035, 1987), electroporation (Neumann et al, EMBOJ 1(7): 841-845, 1982), lipofection (Feigner et al, Proc. Natl. Acad.
  • the ABCA8 cDNA sequence, or fragments thereof can be introduced by infection with virus vectors.
  • Systems are developed that use, for example, retroviruses (Huszar, et al, Proc. Natl.
  • eukaryotic expression systems can be used for studies of ABCA8 encoding nucleic acids and mutant forms of these molecules. Regulatory elements located in the 5' region ofthe ABCA8 gene on genomic clones can be isolated from human genomic DNA libraries using the information contained herein. In other embodiments, the eukaryotic expression systems also may be used to study the function ofthe normal complete protein, specific portions ofthe protein, or of naturally occurring or artificially produced mutant proteins.
  • expression vectors containing the ABCA8 gene sequence or cDNA can be introduced into human cells, mammalian cells from other species or non-mammalian cells, as desired.
  • the choice of cell is determined by the pu ⁇ ose ofthe treatment.
  • monkey COS cells Gluzman, Ce/723:175-182, 1981
  • Chinese hamster ovary CHO
  • mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts are used.
  • a "naked" ABCA8 polynucleotide is placed under the control of appropriate regulatory sequences, and is introduced by local injection at the level ofthe chosen tissue, for example myocardial tissue, the "naked” polynucleotide being absorbed by the myocytes of this tissue.
  • tissue for example myocardial tissue
  • Compositions for use in vitro and in vivo including "naked" polynucleotides are for example described in published international Application WO 95/1 1307 as well as in Tacson et al, Nature Medicine, 2(8):888-892 (1996) and Huygen et al., Nature Medicine, 2(8):893-898 (1996).
  • composition for the in vivo production of ABCA8 protein.
  • This composition includes a polynucleotide encoding the ABCA8 polypeptide placed under the control of appropriate regulatory sequences, in solution in a physiologically acceptable vector.
  • the quantity of vector that is injected into a host organism varies according to the site ofthe injection. As a guide, there may be injected between about 0.1 and about 100 ⁇ g of polynucleotide encoding an ABCA8 protein or variant or gragment thereof into the body of an animal, for instance into a patient likely to develop a disease-linked ABCA8 deficiency.
  • Embodiments described herein thus encompass recombinant vectors that comprise all or part of an ABCA8 encoding sequence, such as the ABCA8 gene or cDNAs or variants thereof, for expression in a suitable host.
  • an ABCA8 DNA is operatively linked in a vector to an expression control sequence in the recombinant DNA molecule so that the ABCA8 polypeptide can be expressed.
  • the expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.
  • the expression control sequence may be specifically selected from the group consisting ofthe lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter ofthe yeast alpha-mating factors and combinations thereof.
  • the host cell which may be transfected with a vector, may be selected from the group consisting of E. coli, Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus or other bacilli; other bacteria; yeast; fungi; insect; mouse or other animal; or plant hosts; or human tissue cells.
  • a diseased condition is in some instances attributed to overexpression of a protein (e.g., HER-2/neu is overexpressed in breast cancer, see Kaya et al., Pathol. Oncol. Res. 7(4): 279-283, 2001).
  • reversal ofthe over-expression e.g., suppression
  • suppression of ABCA8 is achieved in a subject having a transporter malfunction due to overexpression of ABCA8.
  • Reduction of ABCA8 protein expression in a transgenic cell may be obtained for instance by introducing into cells an antisense construct based on the ABCA8 encoding sequence (SEQ ID NO: 1) or gene sequence or flanking regions thereof.
  • a nucleotide sequence from an ABCA8 encoding sequence e.g. SEQ ID NO: 1
  • Other aspects ofthe vector may be chosen as discussed above (see Section VII).
  • the introduced sequence need not be the full-length human ABCA8 cDNA (SEQ ID NO: 1) or gene, and need not be exactly homologous to the equivalent sequence found in the cell type to be transfected. Generally, however, where the introduced sequence is of shorter length, a higher degree of identity to the native ABCA8 sequence will be needed for effective antisense suppression.
  • the introduced antisense sequence in the vector may be at least 15 nucleotides in length, and improved antisense suppression typically will be observed as the length ofthe antisense sequence increases.
  • the length ofthe antisense sequence in the vector advantageously may be greater than 100 nucleotides, and can be up to about the full length ofthe human ABCA8 cDNA or gene.
  • transcription of an antisense construct results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous ABCA8 gene in the cell.
  • antisense RNA molecules bind to the endogenous mRNA molecules and thereby inhibit translation ofthe endogenous mRNA.
  • Expression of ABCA8 can also be reduced using small inhibitory RNAs (siRNAs), for instance using techniques similar to those described previously (see, e.g., Tuschl et ⁇ l., Genes Dev 13, 3191-3197, 1999; Caplen e/ ⁇ /., Proc. N ⁇ t.l Ac ⁇ d. Sci. U. S. A. 98, 9742-9747, 2001 ; and Elbashir et ⁇ l., Nature 41 1, 494-498, 2001).
  • suppression of endogenous ABCA8 expression can be achieved using ribozymes.
  • Ribozymes are synthetic RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S.
  • the inclusion of ribozyme sequences within antisense RNAs may be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that bind to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.
  • dominant negative mutant forms of ABCA8 may be used to block endogenous ABCA8 activity.
  • Monoclonal or polyclonal antibodies may be produced to the native ABCA8 protein or variant forms of this protein.
  • antibodies raised against an ABCA8 protein would specifically detect the ABCA8 protein. That is, such antibodies would recognize and bind the
  • ABCA8 protein or fragments thereof, and would not substantially recognize or bind to other proteins found in human cells.
  • antibodies against the human ABCA8 protein may recognize ABCA8 from other species (e.g., murine ABCA8), and vice versa.
  • Monoclonal or polyclonal antibodies to the protein can be prepared as follows:
  • Monoclonal antibodies to epitopes of ABCA8 proteins can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-497, 1975) or derivative methods thereof.
  • a mouse is repetitively inoculated with a few micrograms ofthe selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells ofthe spleen isolated. The spleen cells are fused with mouse myeloma cells using polyethylene glycol, and the excess, non-fused, cells destroyed by growth ofthe system on selective media comprising aminopterin (HAT media).
  • HAT media aminopterin
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid ofthe wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Enzymol. 70(A): 419-439, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).
  • Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein (for instance, expressed using a method described herein), which, in one specific, non-limiting embodiment, can be modified to enhance immunogenicity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species.
  • small molecules may tend to be less immunogenic than others and may require the use of carriers and adjuvant, examples of which are known.
  • host animals may vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low titer antisera.
  • a series of small doses (ng level) of antigen administered at multiple intradermal sites may be most reliable.
  • An effective immunization protocol for rabbits can be found in Vaitukaitis et al. (J. Clin. Endocrinol. Metab. 33: 988-991 , 1971).
  • booster injections will be given at regular intervals, and antiserum harvested when antibody titer thereof begins to fall, as determined semi-quantitatively (for example, by double irnmunodiffusion in agar against known concentrations ofthe antigen). See, for example, Ouchterlony et al. (In Handbook of Experimental Immunology, Wier, D.
  • the plateau concentration of antibody is usually in the range of about 0.1 to 0.2 mg/ml of serum (about 12 ⁇ M).
  • Affinity ofthe antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Ch. 42, 1980).
  • a third approach to raising antibodies against ABCA8 proteins is to use synthetic peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence ofthe ABCA8 protein.
  • Polyclonal antibodies can be generated by injecting such peptides into, for instance, rabbits.
  • antibodies may be raised against an ABCA8 protein by subcutaneous injection of a recombinant DNA vector that expresses the ABCA8 protein into laboratory animals, such as mice.
  • delivery ofthe recombinant vector into the animals may be achieved using a hand-held form ofthe Biolistic system (Sanford et al., Particulate Sci. Technol 5:27-37, 1987), as described by Tang et al. (Nature 356: 152-154, 1992).
  • expression vectors suitable for this pwpose may include those that express the ABCA8 encoding sequence under the transcriptional control of either the human ⁇ actin promoter or the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • Antibody preparations prepared according to these protocols are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
  • ABCA8 and its functional variants are believed to participate in the regulation of energy- dependent transport of a wide variety of substrates, including ions, metals, amino acids, lipids, peptides, sugars, vitamins, and steroid hormones across membranes.
  • the lack of expression of ABCA8 in a cell leads to a defect in transporter activity.
  • the administration of an ABCA8 recombinant nucleotide to a subject suffering from a transporter deficiency ameliorates the effects ofthe deficiency.
  • the expression of mutant transporter proteins can yield important information about the importance of each amino acid in the protein as well as the details ofthe mechanism of action of these proteins.
  • Cells may have ABCA8 null mutations, ABCA8 missense mutations, or inactivation of ABCA8.
  • a mutant ABCA8 is expressed in a cell but is incapable of localizing to the correct subcellular location.
  • a mutant ABCA8 is incapable of binding to its intracellular binding partners.
  • a mutation in the upstream regulatory region ofthe ABCA8 gene abrogates the expression ofthe protein.
  • a mutant ABCA8 is incapable of transporting target substances.
  • mutations in the ABCA8 gene that may lead to altered transporter function are not included in the cDNA but rather are located in other regions ofthe ABCA8 gene. Mutations located outside ofthe ORF that encode ABCA8 are not likely to affect the functional activity ofthe protein, but rather are likely to result in altered levels ofthe protein in cells. For example, mutations in the promoter region of the ABCA8 gene may prevent transcription of the gene and therefore lead to the complete absence ofthe ABCA8 protein, or absence of certain transcripts ofthe protein, in the cell. Additionally, mutations within introns in the genomic sequence may also prevent expression ofthe ABCA8 protein or lead to expression of a non-functional form ofthe protein.
  • the intron sequences are removed from the RNA molecule, in a process termed splicing, prior to translation ofthe RNA molecule that results in production ofthe encoded protein.
  • splicing a process termed splicing
  • the cellular enzymes that perform the splicing function recognize sequences around the intron/exon border and in this manner recognize the appropriate splice sites. If a mutation exists within the sequence ofthe intron near and exon/intron junction, the enzymes may not recognize the junction and may fail to remove the intron. If this occurs, the encoded protein will likely be defective.
  • splice site mutations may also lead to defects in transporter activity.
  • knowledge ofthe exon structure and intronic splice site sequences of the ABCA8 gene is required to define the molecular basis of these abnormalities.
  • the provision herein ofthe ABCA8 cDNA sequence enables the cloning ofthe entire ABCA8 gene (including the promoter and other regulatory regions and the intron sequences) and the determination of its nucleotide sequence. With this information in hand, diagnosis of a genetic predisposition to transporter deficiency based on DNA analysis will comprehend all possible mutagenic events at the ABCA8 locus.
  • Compounds that modulate the expression or activity of ABCA8 can be used to regulate transporter activity. For instance, in some cases it may be determined that ABCA8 is expressed at low levels in a subject suffering from a transporter deficiency that arose as the result ofthe inefficient expression of ABCA8. Administration, to the subject, of an agent that up-regulates ABCA8 expression can improve transporter activity or ameliorate the transporter deficiency.
  • Changes in transporter activity can be assessed using a variety of in vitro and in vivo assays. These assays can be used to study the effect of ABCA8 on specific cell types or the effect of particular mutations on ABCA8 transporter activity. By systematically introducing mutant ABCA8 constructs into cells and assessing their ability to transport substances across membranes, the importance of each amino acid for the protein's transporter activity can be determined. In addition, these assays can be used to screen for modulators of ABCA8 activity. The modulators identified in this way can then be used to alter ABCA8 expression in cells in vitro or in vivo.
  • cells expressing wild-type ABCA8 are assessed for their ability to transport substances in vitro, as compared to cells that express a mutant ABCA8.
  • an ABCA8 encoding nucleic acid construct is transfected into cells, and levels ofthe transported substance in these cells can be assayed and compared between wild-type and transfected cells for potential differences.
  • Methods of analysis include, for instance, immobilization of the protein on columns to search for compounds that bind the immobilized protein (see Wainer et al., J. Chromatogr. B. Biomed. Sci. Appl.
  • samples or assays that are treated with a test compound that potentially modulates ABC A8 are compared to control samples that are not treated with the test compound, to examine the extent of modulation.
  • the compounds to be tested are present in the range from 0.1 nM to 10 mM.
  • Control samples (untreated with modulators) are assigned a relative ABCA8 activity value of 100%.
  • inhibition of ABCA8 is achieved when the ABCA8 activity value relative to the control is about 90%.
  • inhibition of ABCA8 is achieved when the ABCA8 activity value relative to the control is about 75%, about 50%, about 25%, or about 5%.
  • activation of ABCA8 is achieved when the ABCA8 activity value relative to the control is about 110% (e.g. 10% more than the control). In other embodiments, activation of ABCA8 is achieved when the ABCA8 activity value relative to the control is about 150%, about 175% or about 200%.
  • the effect of test compounds upon ABCA8 activity can be assessed using the assays described above. Such assays include, but are not limited to, the ability to transport substances across membranes.
  • the compounds tested as modulators of ABCA8 are any small chemical compound, or biological entity, such as a polypeptide, sugar, nucleic acid or lipid.
  • the ability of cells transfected with ABCA8 to transport fluorescent compounds can be assessed (see Gribar et al, J. Membr. Biol.173(3): 203-214, 2000).
  • the modulator is a genetically altered version of ABCA8.
  • the effect of potential modulators on ABCA8 protein or mRNA levels, transcriptional activation or repression of a reporter gene is measured.
  • the ABCA8 sequence information presented herein can be used in the area of genetic testing for predisposition to transporter deficiency owing to defects in ABCA8, such as deletion, duplication or mutation ofthe ABCA8 gene, or a portion thereof.
  • the gene sequence ofthe ABCA8 gene, including intron-exon boundaries is also useful in such diagnostic methods. Whether an individual is carrying mutations in the ABCA8 gene (or a portion thereof), or has a duplication(s) or heterozygous or homozygous deletion(s) ofthe ABCA8 gene, may be detected at the DNA level with the use of a variety of techniques.
  • a biological sample ofthe subject which biological sample contains either DNA or RNA derived from the subject, is assayed for a mutated, duplicated or deleted ABCA8 gene.
  • Suitable biological samples include samples containing genomic DNA or RNA obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.
  • Biological samples can be obtained from normal, healthy subjects or from subjects who are predisposed to or who are suffering from any one of a variety ofthe effects of transporter deficiencies such as, but not limited to, hypercholesterolemia, or defective transport of hydrophobic compounds such as lipids, sterols or fatty acids.
  • the detection in the biological sample of either a mutant ABCA8 gene, a mutant ABCA8 RNA, or a duplicated or homozygously or heterozygously deleted ABCA8 gene may be performed by a number of methodologies, examples of which are discussed below.
  • amplification e.g., polymerase chain reaction amplification
  • RT- PCR reverse transcribed RNA
  • the disclosure includes a method of amplifying a nucleic acid and, more particularly, a nucleic acid including any one of SEQ ID NO: 1 , a fragment containing residues 1927- 2046 of SEQ ID NO: 1, and/or of a complementary nucleotide sequence thereof or a variant thereof, contained in a sample, the method involving the steps of: a) bringing the sample in which the presence of a target nucleic acid is suspected into contact with a pair of nucleotide primers whose hybridization position is located respectively on the 5' side and on the 3' side ofthe region ofthe target nucleic acid whose amplification is sought, in the presence ofthe reagents necessary for the amplification reaction; and b) detecting the amplified nucleic acids (if any).
  • nucleotide primers according to the disclosure are useful in methods of genotyping subjects and/or of genotyping populations, in particular in the context of studies of association between particular allele forms or particular forms of groups of alleles (haplotypes) in subjects and the existence of a particular phenotype (character) in these subjects.
  • primers can be used to detect the predisposition of these subjects to develop diseases linked to a deficiency of cholesterol reverse transport and inflammation signaling lipids, or alternatively the predisposition of these subjects to develop a pathology whose candidate chromosomal region is situated on chromosome 17, for example on the 17q arm, and such as in the 17q24 locus.
  • DNA extracted from a biological sample may be used directly for amplification.
  • Direct amplification from genomic DNA would be appropriate for analysis ofthe entire ABCA8 gene including regulatory sequences located upstream and downstream from the open reading frame, or intron/exon borders. Reviews of direct DNA diagnosis have been presented by Caskey (Science 236:1223-1228, 1989) and by Landegren et al. (Science 242:229-237, 1989).
  • mutation scanning techniques appropriate for detecting unknown mutations within amplicons derived from DNA or cDNA could also be performed. These techniques include direct sequencing (without sequencing), single-strand conformational polymo ⁇ hism analysis (SSCP) (for instance, see Hongyo et al, Nucleic Acids Res. 21 :3637-3642, 1993), chemical cleavage (including HOT cleavage) (Bateman et al, Am. J. Med. Genet. 45:233-240, 1993; reviewed in Ellis et al, Hum. Mutat.
  • SSCP single-strand conformational polymo ⁇ hism analysis
  • DNA diagnostic methods can be designed to specifically detect the most common, or most closely disease-linked, ABCA8 defects.
  • the detection of specific DNA mutations may be achieved by methods such as hybridization using allele specific oligonucleotides (ASOs) (Wallace et al, CSHL Symp. Quant. Biol. 51 :257-261, 1986), direct DNA sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA 81 : 1991 - 1995, 1988), the use of restriction enzymes (Flavell et al, Cell 15:25-41, 1978; Geever et al., 1981), discrimination on the basis of electrophoretic mobility in gels with denaturing reagent (Myers and Maniatis, Cold Spring Harbor Symp. Quant. Biol.
  • ASOs allele specific oligonucleotides
  • Oligonucleotides specific to normal or mutant sequences are chemically synthesized using commercially available machines. These oligonucleotides are optionally labeled radioactively with isotopes (such as 32 P) or non-radioactively, with tags such as biotin (Ward and Langer, Proc. Natl. Acad. Sci. USA 78:6633-6657, 1981), and hybridized to individual DNA samples immobilized on membranes or other solid supports by dot-blot or transfer from gels after electrophoresis.
  • isotopes such as 32 P
  • tags such as biotin (Ward and Langer, Proc. Natl. Acad. Sci. USA 78:6633-6657, 1981)
  • Sequence differences between normal and mutant forms ofthe ABCA8 gene may also be revealed by the direct DNA sequencing method of Church and Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1988). Cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined in vitro with nucleic acid amplification, e.g., PCR (Wrichnik et al, Nucleic Acids Res. 15:529-542, 1987; Wong et al, Nature 330:384-386, 1987; Stoflet et al, Science 239:491-494, 1988).
  • a sequencing primer that lies within the amplified sequence is used with double-stranded PCR product or single- stranded template generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent tags.
  • Nucleotide probes and primers hybridizing with an ABCA8 nucleic acid are encompassed by the disclosure, and are suitable for use with the methods discussed above.
  • a nucleotide primer or probe may be prepared by any suitable method well known to persons skilled in the art, including by cloning and action of restriction enzymes or by direct chemical synthesis according to techniques such as the phosphodiester method by Narang et al.
  • Each ofthe nucleic acids according to the disclosure may be labeled, if desired, by inco ⁇ orating a marker that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means.
  • markers may consist of radioactive isotopes (such as 32 P, 33 P, 3 H, 35 S), fluorescent molecules (such as 5-bromodeoxyuridine, fluorescein, acetylaminofluorene, digoxigenin) or ligands such as biotin.
  • the labeling ofthe probes can be carried out for instance by inco ⁇ orating labeled molecules into the polynucleotides by primer extension, or alternatively by addition to the 5' or 3' ends. Examples of nonradioactive labeling of nucleic acid fragments are described in particular in French patent
  • Nucleotide probes and primers according to embodiments ofthe disclosure may have structural characteristics ofthe type to allow amplification ofthe signal, such as the probes described by Urdea et ⁇ t. (Nucleic Acids Symp Ser., 24:197-200, 1991) or alternatively in European patent No. EP-0,225,807.
  • Oligonucleotides according to the disclosure may be used in particular in Southern-type hybridizations with the genomic DNA or alternatively in northern-type hybridizations with the corresponding messenger RNA when the expression ofthe corresponding transcript is sought in a sample, as described herein.
  • the probes and primers according to the disclosure may also be used for the detection of products of in vitro amplification or alternatively for the detection of mismatches.
  • Nucleotide probes or primers according to the disclosure may be immobilized on a solid support.
  • Such solid supports are well known to persons skilled in the art and comprise surfaces of wells of microtiter plates, polystyrene beads, magnetic beads, nitrocellulose bands or microparticles such as latex particles as well as glass or polycarbonate slides or various substances.
  • the probes according to the disclosure may be ordered into matrices such as "DNA chips.” Such ordered matrices have in particular been described in US patent No. 5,143,854, and in published PCT applications WO 90/15070 and WO 92/10092. Support matrices on which oligonucleotide probes have been immobilized at a high density are for example described in US Patent No. 5,412,087 and in published PCT application WO 95/1 1995.
  • nucleic acid fragments derived from a polynucleotide including SEQ ID NO: 1 or a complementary nucleotide sequence, or including residues 1927-2046 of SEQ ID NO: 1, are useful as probes for the detection ofthe presence of at least one copy of a nucleotide sequence ofthe ABCA8 gene or of a fragment or of a variant (e.g., containing a mutation or a polymo ⁇ hism) thereof in a sample.
  • the nucleotide probes or primers according to the disclosure comprise a contiguous nucleotide sequence found in SEQ ID NO: 1, residues 1927-2046 of SEQ ID NO: 1, or a complementary nucleotide thereof.
  • nucleotide probes or primers that include at least 8 consecutive nucleotides having a sequence as shown in residues 1927- 2046 of SEQ ID NO: 1 or a complementary nucleotide sequence thereof.
  • Certain provided nucleotide probes or primers have a length of 10, 12, 15, 18 or 20, to 25, 35, 40, 50, 70, 80, 100 consecutive nucleotides or more of a nucleic acid, which comprise a particular sequence as found in residues 1927-2046 of SEQ ID NO: 1 or a complementary nucleotide sequence thereof.
  • Nucleotide probes or primers also include oligonucleotides that hybridize, under the high stringency hybridization conditions as discussed herein, with a nucleic acid composing SEQ ID NO: 1, residues 1927-2046 of SEQ ID NO: 1, or a complementary nucleotide sequence thereof.
  • the present disclosure also relates to a method of detecting the presence of a nucleic acid including a nucleotide sequence of any one of SEQ ID NO: 1, a fragment containing residues 1927- 2046 of SEQ ID NO: 1 , and/or of a complementary nucleotide sequence thereof, or a nucleic acid fragment or variant of any one of SEQ ID NO: 1, a fragment containing residues 1927-2046 of SEQ ID NO: 1, and/or of a complementary nucleotide sequence thereof in a sample, which method involves: 1) bringing one or more nucleotide probes or primers into contact with the sample to be tested; and 2) detecting the complex that may have formed between the probe(s) and the nucleic acid present in the sample.
  • the oligonucleotide probes and/or primers are immobilized on a support. In some embodiments, the oligonucleotide probes and/or primers include a detectable marker.
  • the nucleotide primers according to the disclosure may be used to amplify any one ofthe nucleic acids according to the disclosure, and more particularly a nucleic acid including a nucleotide sequence of any one of SEQ ID NO: 1, a fragment containing residues 1927-2046 of SEQ ID NO: 1, and/or of a complementary nucleotide sequence thereof.
  • the nucleotide primers according to the disclosure may be used to amplify a nucleic acid fragment or variant of any one of SEQ ID NO: 1, a fragment containing residues 1927-2046 of SEQ ID NO: 1, and/or of a complementary nucleotide sequence thereof.
  • Amplification in this manner may be used to detect a target nucleic acid of interest or alternatively to detect mutations in the coding regions or the non- coding regions ofthe nucleic acids according to the disclosure. Sequence alterations may occasionally generate fortuitous restriction enzyme recognition sites or may eliminate existing restriction sites. Changes in restriction sites are revealed by the use of appropriate enzyme digestion followed by conventional gel-blot hybridization (Southern, J. Mol. Biol. 98:503-517, 1975). DNA fragments carrying the restriction site (either normal or mutant) are detected by their reduction in size or increase in corresponding restriction fragment numbers. Genomic DNA samples may also be amplified by PCR prior to treatment with the appropriate restriction enzyme; fragments of different sizes are then visualized under UV light in the presence of ethidium bromide after gel electrophoresis.
  • Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing reagent. Small sequence deletions and insertions can be visualized by high-resolution gel electrophoresis. For example, a PCR product with small deletions is clearly distinguishable from a normal sequence on an 8 % non-denaturing polyacrylamide gel (WO 91/10734; Nagamine et al, Am. J. Hum. Genet. 45:337- 339, 1989).
  • DNA fragments of different sequence compositions may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific "partial-melting" temperatures (Myers et al, Science 230:1242-1246, 1985).
  • a method of detecting a mutation comprising a single base substitution or other small change could be based on differential primer length in a PCR.
  • an invariant primer could be used in addition to a primer specific for a mutation.
  • the PCR products ofthe normal and mutant genes can then be differentially detected in acrylamide gels.
  • SSCP single-strand conformation polymo ⁇ hism
  • a variety of detection methods such as autoradiography involving radioisotopes, direct detection of radioactive decay (in the presence or absence of scintillant), specfrophotometry involving calorigenic reactions and fluorometry involved fluorogenic reactions, may be used to identify specific individual genotypes.
  • a system capable of detecting such multiple mutations likely will be desirable.
  • a nucleic acid amplification reaction with multiple, specific oligonucleotide primers and hybridization probes may be used to identify all possible mutations at the same time (Chamberlain et al, Nucl. Acids Res. 16:1141-1155, 1988).
  • the procedure may involve immobilized sequence-specific oligonucleotide probes (Saiki et al, Proc. Nat. Acad. Sci. USA 86:6230-6234, 1989).
  • Expression levels ofthe ABCA8 gene can also be determined by methods such as Northern or Southern blot analysis using labeled oligonucleotides specific to normal or mutant sequences. These oligonucleotides are labeled radioactively with isotopes (such as 32 P) or non-radioactively, with tags such as biotin (Ward and Langer, Proc. Natl. Acad. Sci. USA 78: 6633-6657, 1981), and hybridized to individual DNA samples immobilized on membranes or other solid supports by dot-blot or transfer from gels after electrophoresis.
  • isotopes such as 32 P
  • tags such as biotin
  • Quantitative or semi-quantitative PCR can also be used to measure the amount of ABCA8 cDNA in a sample using ABCA8 oligonucleotide primers.
  • Visualization methods such as autoradiography or fluorometric (Landegren et al, Science 242: 229- 237, 1989) or colorimetric reactions (Gebeyehu e/ al, Nucleic Acids Res. 15: 4513-4534, 1987) can be used to detect a signal and the signals quantitated using, for instance, a spectrophotometer, a scintillation counter, a densitometer or a Phosphorimager (Amersham Biosciences).
  • the Phosphorimager is able to analyze both DNA and protein samples from blots and gels using autoradiographic, direct fluorescence or chemifluorescence detection. Because the Phosphorimager is more sensitive than ordinary x-ray film, exposure times can be reduced up to ten-fold and signal quantitation of both weak and strong signals on the same blot is possible. Images can be visualized and evaluated with the aid of computer programs such as ImageQuantTM.
  • the ABCA8 sequence information presented herein is useful in detecting the presence or absence of ABCA8 in cultured cells or primary cells. Quantitative and qualitative methods of detection of proteins are well-known in the art, and are discussed herein. Quantitative detection is useful for diagnosing over- or underexpression of ABCA8 proteins in a subject, while qualitative information gives, for example, information regarding tissue types in which ABCA8 may be expressed. For such qualitative or quantitative assessment, a biological sample ofthe subject, which biological sample contains either DNA or RNA derived from the subject, is assayed for the presence or absence of ABCA8.
  • Suitable biological samples include samples containing genomic DNA or RNA obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.
  • Biological samples can be obtained from normal, healthy subjects or from subjects who are predisposed to or who are suffering from any one of a variety ofthe effects of transporter deficiencies such as, but not limited to, hypercholesterolemia, or defective transport of hydrophobic compounds such as lipids, sterols or fatty acids.
  • Antibodies can be used to assess the presence or absence of ABCA8 proteins in cultured cells or primary cells.
  • the determination whether an antibody specifically detects ABCA8 proteins is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al, In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989).
  • it is determined whether a given antibody preparation (such as one produced in a mouse) specifically detects ABCA8 proteins by Western blotting.
  • total cellular protein is extracted from human cells (for example, lymphocytes) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel.
  • the cellular protein is extracted from a biological sample.
  • the proteins are then transferred to a membrane (for example, nitrocellulose or PVDF) by Western blotting, and the antibody preparation is incubated with the membrane.
  • a membrane for example, nitrocellulose or PVDF
  • the presence of specifically bound antibodies is detected by the use of (by way of example) an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase.
  • an enzyme such as alkaline phosphatase.
  • Application of an alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immunolocalized alkaline phosphatase.
  • Antibodies that specifically detect ABCA8 proteins will, by this technique, be shown to bind to ABCA8 protein bands (which will be localized at a given position on the gel determined by its molecular weight). Non-specific binding ofthe antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-ABCA8 protein binding.
  • substantially pure ABCA8 protein suitable for use as an immunogen is isolated from the transfected cells as described above.
  • concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon (Millipore, Bedford, Massachusetts) or similar filter device, to the level of a few micrograms per milliliter.
  • antibodies against ABCA8 proteins are used to localize ABCA8 to specific cell types or to specific subcellular locations in immunohistochemical or immunofluorescence assays.
  • the cells are selected from a variety of cell lines.
  • primary cells are isolated from a subject and are maintained in culture or the sample is sectioned and the sections are prepared directly for immunohistochemistry or immunofluorescence.
  • the cells are fixed, incubated in a blocking medium, incubated with the antibody directed against ABCA8 followed by a second incubation with a secondary antibody that is conjugated to a fluorescent probe or a colorimetric agent.
  • Cells that express an ABCA8 protein that is recognized by the antibody exhibit a color or are fluorescent when viewed under a light or fluorescence microscope, respectively.
  • An alternative method of diagnosing ABCA8 gene deletion, amplification, or mutation is to quantitate the level of ABCA8 protein in the cells of a subject.
  • this diagnostic tool would be useful for detecting reduced levels of ABCA8 protein that result from, for example, mutations in the promoter regions ofthe ABCA8 gene or mutations within the coding region ofthe gene that produce truncated, non-functional or unstable polypeptides, as well as from deletions ofthe entire ABCA8 gene.
  • duplications ofthe ABCA8 gene may be detected as an increase in the expression level of this protein.
  • the determination of reduced or increased ABCA8 protein levels would be an alternative or supplemental approach to the direct determination of ABCA8 gene deletion, duplication or mutation status by the methods outlined above.
  • the availability of antibodies specific to ABCA8 proteins will facilitate the quantitation of cellular ABCA8 proteins by one of a number of immunoassay methods, which are well known in the art and are presented herein and in, for instance, Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). Many techniques are commonly known in the art for the detection and quantification of antigen.
  • the purified antigen will be bound to a substrate, the antibody ofthe sample will bind via its Fab portion to this antigen, the substrate will then be washed and a second, labeled antibody will then be added, which will bind to the Fc portion ofthe antibody that is the subject ofthe assay.
  • the second, labeled antibody will be species specific, i.e., if the serum is from a rabbit, the second, labeled antibody will be anti-rabbit-IgG antibody.
  • the specimen will then be washed and the amount ofthe second, labeled antibody that has been bound will be detected and quantified by standard methods.
  • a biological sample ofthe subject which sample includes cellular proteins, can be used.
  • a biological sample may be obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, amniocentesis samples, surgical specimens and autopsy material.
  • Biological samples can be obtained from normal, healthy subjects or from subjects who are predisposed to or who are already suffering from any one of a variety of transporter deficiencies, such as, but not limited to, defective transport of cholesterol, fatty acids, or lipids, or the effects of defective steroid hormones derived from the cholesterol of individuals with mutant ABCA8.
  • Quantitation of ABCA8 protein can be achieved for instance by immunoassay and compared to levels ofthe protein found in healthy cells (e.g., cells from a subject known not to suffer from a transporter deficiency).
  • a significant (e.g., 10% or greater, for instance, 20%, 25%, 30%>, 50% or more) reduction in the amount of ABCA8 protein in the cells of a subject compared to the amount of ABCA8 protein found in normal human cells would be taken as an indication that the subject may have deletions or mutations in the ABCA8 gene locus, whereas in another embodiment, a significant (e.g., 10% or greater, for instance, 20%, 25%, 30%, 50% or more) increase would indicate that a duplication or enhancing mutation had occurred.
  • Mutant organisms that under-express or over-express ABCA8 proteins are useful for research. Such mutants allow insight into the physiological and/or pathological role of ABCA8 in a healthy and/or pathological organism via study using a mutant model system (e.g., a transgenic ABCA8 knockout mouse). These mutants are "genetically engineered,” meaning that information in the form of nucleotides has been transferred into the mutant's genome at a location, or in a combination, in which it would not normally exist. Nucleotides transferred in this way are said to be “non-native.” In one embodiment, a non-ABCA8 promoter inserted upstream of a native ABCA8 gene would be non-native. In other embodiments, an extra copy of an ABCA8 gene or other encoding sequence on a plasmid, transfected into a cell, would be non-native, whether that extra copy was ABCA8 derived from the same, or a different species.
  • a mutant model system e.g.
  • Mutants may be, for example, produced from mammals, such as mice, that either over- express or under-express ABCA8 protein, or that do not express ABCA8 at all.
  • over-expression mutants are made by increasing the sequences (such as genes) in the organism.
  • over-expression mutants are made by introducing an ABCA8-encoding sequence into the organism under the control of a constitutive or inducible or viral promoter such as the mouse mammary tumor virus (MMTV) promoter or the whey acidic protein (WAP) promoter or the metallothionein promoter.
  • MMTV mouse mammary tumor virus
  • WAP whey acidic protein
  • mutants that under- express ABCA8 may be made by using an inducible or repressible promoter, or by deleting the ABCA8 gene, or by destroying or limiting the function ofthe ABCA8 gene, for instance by disrupting the gene by transposon insertion.
  • antisense genes may be engineered into the organism, under a constitutive or inducible promoter, to decrease or prevent ABCA8 expression, as discussed above.
  • a gene is "functionally deleted" when genetic engineering has been used to negate or reduce gene expression to negligible levels.
  • a mutant is referred to in this application as having the ABCA8 gene altered or functionally deleted, this refers to the ABCA8 gene and to any ortholog of this gene.
  • a mutant is referred to as having "more than the normal copy number" of a gene, this means that it has more than the usual number of genes found in the wild-type organism, e.g., in the diploid mouse or human.
  • a mutant mouse over-expressing ABCA8 may be made by constructing a plasmid having the ABCA8 gene driven by a promoter, such as the mouse mammary tumor virus (MMTV) promoter or the whey acidic protein (WAP) promoter.
  • MMTV mouse mammary tumor virus
  • WAP whey acidic protein
  • this plasmid may be introduced into mouse oocytes by microinjection. The oocytes are implanted into pseudopregnant females, and the litters are assayed for insertion ofthe transgene. Multiple strains containing the transgene are then available for study.
  • WAP is quite specific for mammary gland expression during lactation, and MMTV is expressed in a variety of tissues including mammary gland, salivary gland and lymphoid tissues.
  • MMTV is expressed in a variety of tissues including mammary gland, salivary gland and lymphoid tissues.
  • other promoters might be used to achieve various patterns of expression, e.g., the metallothionein promoter.
  • an inducible system may be created in which the subject expression construct is driven by a promoter regulated by an agent that can be fed to the mouse, such as tetracycline.
  • an agent that can be fed to the mouse, such as tetracycline.
  • a mutant knockout animal from which the ABCA8 gene is deleted or otherwise disabled can be made by removing coding regions ofthe ABCA8 gene from embryonic stem cells.
  • the methods of creating deletion mutations by using a targeting vector have been described (see, for instance, Thomas and Capecch, Cell 51: 503-512, 1987).
  • knockout mice are used as hosts to test the effects of various ABCA8 constructs on cell growth.
  • transgenic mice with the endogenous ABCA8 gene knocked-out can be used in an assay to screen for compounds that modulate the ABCA8 activity.
  • a transgenic mouse that is heterozygous or homozygous for integrated transgenes that have functionally disrupted the endogenous ABCA8 gene can be used as a sensitive in vivo screening assay for the ABCA8 ligands and modulators of ABCA8 activity.
  • retroviruses are a preferred vector for experiments in medical genetics, as they yield a high efficiency of infection and stable integration and expression (Orkin et al, Prog. Med. Genet. 7:130-142, 1988).
  • the full-length ABCA8 gene or cDNA can be cloned into a retroviral vector and driven from either its endogenous promoter or, for instance, from the retroviral LTR (long terminal repeat).
  • viral transfection systems may also be utilized for this type of approach, including adenovirus, adeno- associated virus (AAV) (McLaughlin et al, J. Virol.
  • Vaccinia virus Moss et al, Annu. Rev. Immunol. 5:305-324, 1987
  • Bovine Papilloma virus Rosmussen et al, Methods Enzymol. 139:642-654, 1987
  • members ofthe he ⁇ esvirus group such as Epstein-Barr virus (Margolskee et al, Mol. Cell. Biol. 8:2837-2847, 1988).
  • RNA-DNA hybrid oligonucleotides as described by Cole-Strauss et al. (Science 273:1386-1389, 1996). This technique may allow for site- specific integration of cloned sequences, thereby permitting accurately targeted gene replacement.
  • lipidic and liposome-mediated gene delivery will be used for transfection of various genes (for reviews, see Templeton and Lasic, Mol. Biotechnol. 11:175-180, 1999; Lee and Huang, Crit. Rev. Ther. Drug Carrier Syst. 14:173-206; and Cooper, Semin. Oncol. 23:172-187, 1996).
  • cationic liposomes will be used as a viable alternative to the viral vectors (de Lima et al, Mol Membr. Biol. 16: 103-109, 1999).
  • cationic liposomes can be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al, Cancer Gene Ther. 3:250-256, 1996).
  • a pharmaceutical composition intended for the prevention of or treatment of a patient or a subject affected with ABCA8 deficiency, including a recombinant vector according to the disclosure, in combination with one or more physiologically compatible excipients.
  • the disclosure also relates to the use of a nucleic acid according to the disclosure, encoding the ABCA8 protein or a variant or fragment thereof, for the manufacture of a medicament intended for the prevention of a disease, for instance atherosclerosis or arteriosclerosis in various forms or more particularly for the treatment of subjects affected by a dysfunction of cholesterol reverse transport or inflammatory lipophilic substances transport.
  • the disclosure also relates to the use of a recombinant vector according to the disclosure, including an ABCA8 nucleic acid, for the manufacture of a medicament intended for the prevention of a disease, such as atherosclerosis or arteriosclerosis in various forms, or in some embodiments particularly for the treatment of subjects affected by a dysfunction of cholesterol reverse transport or inflammatory lipophilic substances transport.
  • a disease such as atherosclerosis or arteriosclerosis in various forms, or in some embodiments particularly for the treatment of subjects affected by a dysfunction of cholesterol reverse transport or inflammatory lipophilic substances transport.
  • the present disclosure also relates to a pharmaceutical composition including one or more defective recombinant viruses as described above.
  • compositions disclosed herein may be formulated for administration by the topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular or transdermal route and so forth.
  • the pharmaceutical compositions comprise a pharmaceutically acceptable vehicle or physiologically compatible excipient for an injectable formulation, in particular for an intravenous injection, such as for example into the subject's portal vein.
  • injectable formulation in particular for an intravenous injection, such as for example into the subject's portal vein.
  • the disclosure further relates to various methods of screening compounds or small molecules for therapeutic use that are useful in the treatment of diseases caused by a deficiency in ABCA8, further in the transport of cholesterol, or more particularly, in inflammatory lipid substances.
  • the disclosure therefore also relates to the use ofthe ABCA8 polypeptide, or of cells expressing the ABCA8 polypeptide, for screening active ingredients for the prevention and/or treatment of diseases resulting from a dysfunction in ABCA8 deficiency.
  • Peptides from functional sites for instance, from within ABC trans-membrane and nucleotide-binding domains as described herein
  • oligopeptide or immunogenic fragments of ABCA8 for instance oligopeptides within the region encompassed by residues 597-636 of SEQ ID NO: 2
  • the polypeptide fragment(s) used in this type of screening may be free in solution, bound to a solid support, at the cell surface, or in the cell. The formation ofthe binding complexes between the ABCA8 polypeptide fragment and the tested agent can then be measured.
  • the antibodies may be used to detect the presence of a peptide having a common antigenic unit with an ABCA8 polypeptide or protein.
  • this disclosure relates to the use of any method of screening products (i.e., compounds, small molecules, and so forth), based on translocation of a substrate, such as cholesterol or a lipophilic substance, between membranes or vesicles.
  • a substrate such as cholesterol or a lipophilic substance
  • translocation can be ofthe synthetic or cellular type (e.g., mammal cells, insect cells, bacteria, or yeasts expressing constitutively or having inco ⁇ orated human ABCA8 encoding nucleic acids).
  • labeled lipophilic substance analogs may be used.
  • the disclosure also relates to a method of screening a compound, or small molecule active or potentially active in the transport of lipophilic substances, for activity as an agonist or antagonist of an ABCA8 polypeptide, the method including the following: a) preparing a membrane vesicle including an ABCA8 polypeptide(s) and a substrate, which substrate optionally includes a detectable marker; b) incubating the vesicle with a candidate agonist or antagonist compound; c) qualitatively and/or quantitatively measuring release ofthe substrate from the vesicles; and d) comparing the release measurement with a measurement of release of labeled substrate by a vesicle that has not been previously incubated with the agonist or antagonist candidate compound.
  • a membrane vesicle can be a synthetic lipid vesicle, which may be prepared according to techniques well known to a person skilled in the art.
  • the ABCA8 protein is recombinant protein.
  • the membrane vesicle is a vesicle of a plasma membrane derived from cells expressing the ABCA8 polypeptide. These may be cells naturally expressing the ABCA8 polypeptide or cells transfected with a nucleic acid encoding an ABCA8 polypeptide or recombinant vector including a nucleic acid encoding an ABCA8 polypeptide.
  • the substrates are lipid substrates.
  • Lipid substrates used in the above screening method may include (but are not limited to) prostaglandins or prostacyclins, cholesterol or phosphatidylcholine, radioactively labeled lipid substrates (e.g., with an isotope chosen from 3 H or l25 I), and fluorescently labeled compounds (e.g., labeled with NBD or pyrene).
  • the measurement ofthe fluorescence or ofthe radioactivity released by the vesicle is a direct reflection ofthe activity of substrate transport by the ABCA8 polypeptide.
  • the membrane vesicle including the labeled substances and the ABCA8 polypeptide can be immobilized at the surface of a solid support prior to incubating them with the candidate compound.
  • the disclosure also relates to a method of screening a compound or small molecule active (or thought to potentially be active) on the transport of cholesterol or lipid substances, an agonist or antagonist ofthe ABCA8 polypeptide, the method including the following: a) obtaining cells, for example a cell line that, either naturally or after transfecting the cell with a ABCA8 encoding nucleic acid, expresses the ABCA8 polypeptide; b) incubating the cells of a) in the presence of an anion labeled with a detectable marker; c) washing the cells of b) in order to remove the excess ofthe labeled anion which has not penetrated into these cells; d) incubating the cells obtained in c) with an agonist or antagonist candidate compound for the ABCA8 polypeptide; e) measuring efflux ofthe labeled anion; and f) comparing the value of efflux ofthe labeled anion determined in e) with a value ofthe efflux of a labeled anion measured with cells that
  • the cells used in the screening method described above may be cells not naturally expressing, or alternatively expressing at a low level, the ABCA8 polypeptide, the cells being transfected with a recombinant vector according to the disclosure capable of directing the expression of a nucleic acid encoding the ABCA8 polypeptide.
  • cells may be used that have a natural deficiency in anion transport, or are pretreated with one or more anion channel inhibitors such as VerapamilTM or tetraethylammonium.
  • the anion used may be a radioactively labeled iodide, such as the salts K 125 I or Na 125 I.
  • the value of efflux ofthe labeled anion may be determined by measuring the quantity of labeled anion present at a given time in the cell culture supernatant.
  • the value of efflux ofthe labeled anion may be determined as the proportion of radioactivity found in the cell culture supernatant relative to the total radioactivity corresponding to the sum ofthe radioactivity found in the cell lysate and the radioactivity found in the cell culture supernatant.
  • the cells used belong to the human or mouse myocytes.
  • the compound used to stimulate the production of interleukin may a lipopolysaccharide.
  • the production of all interleukins and TNF alpha by the cells and the level of expression ofthe messenger RNA encoding interleukin may be qualitatively and/or quantitatively determined.
  • Kits which contain the necessary reagents for determining ABCA8 gene copy number, for determining altered expression of ABCA8 mRNA or ABCA8 protein, or for detecting polymo ⁇ hisms in ABCA8 alleles.
  • Instructions provided in the diagnostic kits can include calibration curves, diagrams, illustrations, or charts or the like to compare with the determined (e.g., experimentally measured) values or other results.
  • Kits are also provided that contain cells that serve as either positive or negative controls. These control cells can be compared to experimental samples containing similar cells, for instance cells of unknown gene activity, mutational state, protein expression level, and so forth.
  • the nucleotide sequences disclosed herein, and fragments thereof, can be supplied in the form of a kit for use in detection of ABCA8 genomic sequences, for instance in order to diagnose a deficiency in transporter activity.
  • a kit for use in detection of ABCA8 genomic sequences for instance in order to diagnose a deficiency in transporter activity.
  • an appropriate amount of one or more ofthe ABCA8-specif ⁇ c oligonucleotide primers is provided in one or more containers.
  • the oligonucleotide primers may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance.
  • the containers) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles.
  • pairs of primers may be provided in pre-measured single use amounts in individual, typically disposable, tubes or equivalent containers.
  • the sample to be tested for the presence o ABCA8 genomic amplification can be added to the individual tubes and in vitro amplification carried out directly.
  • each oligonucleotide primer supplied in the kit can be any appropriate amount, depending for instance on the market to which the product is directed.
  • the kit is adapted for research or clinical use and the amount of each oligonucleotide primer provided is an amount sufficient to prime several in vitro amplification reactions.
  • Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction. General guidelines may for instance be found in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990), Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989), and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
  • a kit may include more than two primers, in order to facilitate the PCR in vitro amplification of ABCA8 sequences, for instance the ABCA8 gene, specific exon(s) or other portions ofthe gene, or the 5' or 3' flanking region thereof.
  • a kit may include primers for amplification of a nucleotide binding domain (e.g. residues 502-656 or 1308-1621 of SEQ ID NO: 2).
  • kits may also include the reagents necessary to carry out PCR in vitro amplification reactions, including, for instance, DNA sample preparation reagents, appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs). Instructions may also be included.
  • appropriate buffers e.g., polymerase buffer
  • salts e.g., magnesium chloride
  • dNTPs deoxyribonucleotides
  • kits may include either labeled or unlabeled oligonucleotide probes for use in detection ofthe in vitro amplified ABCA8 sequences.
  • the appropriate sequences for such a probe will be any sequence that falls between the annealing sites ofthe two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the in vitro amplification reaction.
  • the kit provides one or more control sequences for use in the amplification reactions.
  • the design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
  • Kits similar to those disclosed above for the detection of ABCA8 genomic sequences can be used to detect ABCA8 mRNA expression levels.
  • One embodiment of such a kit includes an appropriate amount of one or more ofthe oligonucleotide primers for use in reverse transcription amplification reactions, similarly to those provided above, with art-obvious modifications for use with RNA.
  • kits for detection of ABCA8 mRNA expression levels may also include the reagents necessary to carry out RT-PCR in vitro amplification reactions, including, for instance, RNA sample preparation reagents (including e.g., an RNAse inhibitor), appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs). Instructions also may be included.
  • RNA sample preparation reagents including e.g., an RNAse inhibitor
  • appropriate buffers e.g., polymerase buffer
  • salts e.g., magnesium chloride
  • dNTPs deoxyribonucleotides
  • kits may include either labeled or unlabeled oligonucleotide probes for use in detection ofthe in vitro amplified target sequences.
  • the appropriate sequences for such a probe will be any sequence that falls between the annealing sites ofthe two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the PCR reaction.
  • the kit provides one or more control sequences for use in the RT- PCR reactions.
  • the design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art.
  • kits may be provided with the necessary reagents to carry out quantitative or semi-quantitative Northern analysis of ABCA8 mRNA.
  • kits include, for instance, at least one ⁇ C S-specific oligonucleotide for use as a probe.
  • This oligonucleotide may be labeled in any conventional way, including with a selected radioactive isotope, enzyme substrate, co-factor, ligand, chemiluminescent or fluorescent agent, hapten, or enzyme.
  • kits for the detection of ABCA8 protein expression include for instance at least one target protein specific binding agent (e.g., a polyclonal or monoclonal antibody or antibody fragment) and may include at least one control.
  • the ABCA8 protein specific binding agent and control may be contained in separate containers.
  • the kits may also include means for detecting ABCA8:agent complexes, for instance the agent may be detectably labeled. If the detectable agent is not labeled, it may be detected by second antibodies, or protein A for example, which may also be provided in some kits in one or more separate containers. Such techniques are well known.
  • kits include instructions for carrying out the assay. Instructions will allow the tester to determine whether ABCA8 expression levels are altered, for instance in comparison to a control sample.
  • reaction vessels and auxiliary reagents such as cells, chromogens, buffers, media, enzymes, etc. also may be included in the kits.
  • an effective and convenient immunoassay kit such as an enzyme-linked immunosorbant assay can be constructed to test anti-ABCA8 antibody in human serum, as reported for detection of non-specific anti-ovarian antibodies (Wheatcroft et al, Clin. Exp. Immunol. 96:122-128, 1994; Wheatcroft et al, Hum. Reprod. 12:2617-2622, 1997).
  • expression vectors can be constructed using the human ABCA8 cDNA to produce the recombinant human ABCA8 protein in either bacteria or baculovirus (as described herein).
  • affinity purification is used to generate unlimited amounts of pure recombinant ABCA8 protein.
  • an assay kit could provide the recombinant protein as an antigen and enzyme-conjugated goat anti-human IgG as a second antibody as well as the enzymatic substrates. Such kits can be used to test if the sera from a subject contain antibodies against ABCA8.
  • kits for Detection ofHomozv ⁇ ous versus Heterozv ⁇ ous Allelism
  • kits that allow differentiation between individuals who are homozygous versus heterozygous for a polymo ⁇ hism of ABCA8.
  • such kits provide the materials necessary to perform oligonucleotide ligation assays (OLA), for instance as described at Nickerson et al. (Proc. Natl. Acad. Sci. USA 87:8923-8927, 1990).
  • these kits contain one or more microtiter plate assays, designed to detect allelism in the ABCA8 sequence of a subject, as described herein.
  • additional components in some of these kits may include instructions for carrying out the assay. Instructions will allow the tester to determine whether an ABCA8 allele is homozygous or heterozygous.
  • reaction vessels and auxiliary reagents such as chromogens, buffers, enzymes, etc. may also be included in the kits.
  • the kit may provide one or more control sequences for use in the
  • kits for Identifying Modulators ofABCA8 Activity
  • kits that allow for the identification of modulators of ABCA8 activity.
  • such kits provide the materials necessary to assess the activity of ABCA8 in vitro.
  • this kit contains aliquots of isolated ABCA8 and cultured cells.
  • the kit contains cell lines that express either wildtype or mutant ABCA8.
  • additional components in some of these kits may include instructions for carrying out the assay.
  • reaction vessels and auxiliary reagents such as chromogens, buffers, media, enzymes, etc. may also be included in the kits.
  • This example provides a description of how the ABCA8 genomic sequence was first identified.
  • the genomic DNA analysis was performed by combination of several gene-finding programs such as GENSCAN (Burge and Karlin, J Mol Biol; 268(l):78-94, 1997), FGENEH/FEXH (Solovyev and Salamov, ISMB; 5:294-302, 1997), and XPOUND (Thomas and Skolnick, J Math Appl Med Biol; ⁇ 1(1): 1-16, 1994).
  • GENSCAN Bos Computer Group
  • FGENEH/FEXH Solovyev and Salamov
  • XPOUND Thimas and Skolnick, J Math Appl Med Biol; ⁇ 1(1): 1-16, 1994.
  • the second step in the genomic DNA analysis was homology searching in the EST and protein databases. Combination of software performing database searching and software for exon/intron prediction gave the most sensitive and specific results. Sequence assembly and analysis were performed using the Genetics Computer Group (GCG) sequence analysis software package.
  • GCG Genetics Computer Group
  • NBD corresponds to the extended nucleotide binding domain, i.e. in ABCAl it spans from amino acids 885-1152 for the N-terminal one and 1918-2132 for the C-terminal one.
  • the alignments ofthe TMN and TMC domains ofthe 17q24 genes were used to perform phylogenetic analysis along with ABCAl, ABCA2, ABCA3, ABCA4, and C. elegans ABCA-related gene.
  • the sequences were aligned with PILEUP (Wisconsin Package) and converted to a PHYLIP format.
  • Phylogenetic analysis was performed with the PHYLIP Protpars (maximum parsimony) and Protdist (neighbor joining) methods (PHYLIP package).
  • the Seqboot program was used for bootstrap analysis (100 iterations) and consensus trees were generated with Consense.
  • EXAMPLE 2 Human ABCA8 cDNA.
  • This Example describes the isolation and identification of cDNA molecules encoding the full-length human ABCA8 protein.
  • Reverse transcription In a total volume of 11.5 ⁇ l, 500 ng of mRNA poly(A)+ (prepared using a CLONTECH mRNA purification kit) mixed with 500 ng of oligo-dT were denatured at 70°C for 10 minutes and then chilled on ice. After addition of 10 units of RNasin, 10 mM DTT, 0.5 mM dNTP, Superscript first strand buffer and 200 units of Superscript II (Life Technologies), the reaction was incubated for 45 minutes at 42°C.
  • Each polymerase chain reaction contained 400 ⁇ M each dNTP, 2 units of Thermus aquaticus (Taq) DNA polymerase (Ampli Taq Gold; Perkin Elmer), 0.5 ⁇ M each primer, 2.5 mM MgCl 2 , PCR buffer and 50 ng of DNA, or about 25 ng of cDNA, or 1/50* of a primary PCR mixture. Reactions were carried out for 30 cycles in a Perkin Elmer 9700 thermal cycler in 96-well microtiter plates.
  • each cycle consisted of: a denaturation step of 30 seconds (94°C), a hybridization step of 30 seconds (64°C for 2 cycles, 61°C for 2 cycles, 58°C for 2 cycles and 55°C for 28 cycles), and an elongation step of 1 minute/kb (72°C).
  • PCR amplification ended with a final 72°C extension of 7 minutes.
  • control reactions without reverse transcriptase and reactions containing water instead of cDNA were performed for every sample.
  • Oligonucleotides were selected using Prime from GCG package or Oligo 4 (National Biosciences, Inc.) softwares. Primers were ordered from Life Technologies, Ltd. and used without further purification.
  • PCR products were analyzed and quantified by agarose gel electrophoresis, purified with a P100 column. Purified PCR products were sequenced using ABI Prism BigDye terminator cycle sequencing kit (Perkin Elmer Applied Biosystems). The sequence reaction mixture was purified using Microcon-100 microconcentrators (Amicon, Inc., Beverly). Sequencing reactions were resolved on an ABI 377 DNA sequencer (Perkin Elmer Applied Biosystems) according to manufacturer's protocol (Applied Biosystems, Perkin Elmer).
  • RNA is extracted from human cells by any one of a variety of methods well known to those of ordinary skill in the art.
  • Sambrook et ⁇ l., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, and Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998 provide descriptions of methods for RNA isolation.
  • Any human cell line derived from a nonABCA8 deleted individual would be suitable, such as the widely used HeLa cell line, or the WI-38 human skin fibroblast cell line available from the American Type Culture Collection, Rockville, MD.
  • the extracted RNA is then used as a template for performing the reverse transcription-polymerase chain reaction (RT-PCR) amplification of cDNA.
  • RT-PCR reverse transcription-polymerase chain reaction
  • PCR primers will be made according to the portions ofthe cDNA that are to be amplified. Primers may be chosen to amplify small segments of a cDNA or the entire cDNA molecule. Variations in amplification conditions may be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). By way of example, ⁇ ZJC ⁇ 5-encoding sequences may be amplified theoretically using the following combination of 5' -> 3' primers:
  • An additional set of primers that may be used to amplify an ABCA8 encoding sequence are:
  • primer 3 5' CATGGTAGAGACCAGGAGAAAA 3' (SEQ ID NO: 9)
  • primer 4 5' CTCAGTCATGTGAGCTGTTGC 3' (SEQ ID NO: 10)
  • the coding sequence o ABCA8 may be amplified using the following combination of primers:
  • primer 5 5' ATGAGGAAGAGAAAG 3' (SEQ ID NO: 5)
  • primer 6 5' TTAAGGCTCTTCCTGGGGG 3' (SEQ ID NO: 6).
  • the newly identified portion ofthe ABCA8 cDNA (nucleotides 1927-2046 of SEQ ID NO: 1) may be amplified using the following combination of primers:
  • primer 7 5' ATACAAAGGGTTC 3' (SEQ ID NO: 7)
  • primer 8 5" CTGAGGATCTCC 3' (SEQ ID NO: 8).
  • primers are illustrative only; one skilled in the art will appreciate that many different primers may be derived from the provided cDNA sequence in order to amplify particular regions thereof.
  • the profile of expression of ABCA8 polynucleotides can be determined using art-known techniques, such as PCR-coupled reverse transcription, which has been described for instance by Sambrook et al. (1989, Molecular cloning: A laboratory manual. 2 nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). This example provides representative methods and results from various tissues. RT-PCR
  • a pair of primers as described above may be synthesized corresponding to a human ABCA8 cDNA (SEQ ID NO: 1). This primer pair may be used to detect the corresponding ABCA8 cDNA.
  • the polymerase chain reaction (PCR) was carried out on commercially provided first-strand cDNA templates (CLONTECH) corresponding to retro-transcribed polyA + mRNAs.
  • Reverse transcription to cDNA was carried out with the enzyme SUPERSCRIPT II (GibcoBRL, Life Technologies) according to the conditions described by the manufacturer.
  • the polymerase chain reaction was carried out according to standard conditions, in 20 ⁇ l of reaction mixture with 25 ng of cDNA preparation.
  • the reaction mixture was composed of 400 ⁇ M of each ofthe dNTPs, 2 units of Thermus aquaticus (Taq) DNA polymerase (Ampli Taq Gold; Perkin Elmer), 0.5 ⁇ M of each primer, 2.5 mM MgCl 2 , and PCR buffer.
  • Taq Thermus aquaticus
  • the profile of expression o ABCA8 polynucleotides can be determined using art-known techniques, such as Northern blot analysis, which has been described for instance by Sambrook et al. (1989, Molecular cloning: a laboratory manual. 2 nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). This example provides representative methods.
  • PCR products are gel-purified using Qiaquick® column (Qiagen). 10-20 ng of purified PCR product are radiolabeled with [ ⁇ 32 P]dCTP (Amersham; 6000 Ci/mmol, 10 mCi/ml) by the random priming method (Rediprime kit; Amersham) according to the manufacturer's protocol. Uninco ⁇ orated radioactive nucleotides are separated from the labeled probe by filtration on a G50 microcolumn (Pharmacia). Probe is competed with 50 ⁇ g of denatured human Cotl DNA for 2 hours at 65°C.
  • SSPE 5x Denhardt's, 2.5% Dextran, 0.5% SDS, 50% formamide, 100 ⁇ g/ml denatured salmon sperm DNA, 40 ⁇ g denatured human DNA
  • radiolabeled probe 2.10 6 cp /ml hybridization solution, as described above
  • 40 ⁇ g of denatured human DNA Filters are washed in 2x SSC for 30 minutes at room temperature, twice in 2x SSC-0.1% SDS for 10 minutes at 65°C and twice in lx SSC-0.1% SDS for 10 minutes at 65°C.
  • Northern blots are analyzed after overnight (or other appropriate duration) exposure on, for instance, a Storm system (Molecular Dynamics, Sunnyvale, CA). Human transferrin probe can be used to control the amount of RNA loaded in each lane ofthe membrane.
  • the normal ABCA8 polypeptide (SEQ ID NO: 2) encoded by complete corresponding cDNA (SEQ ID NO: 1) whose isolation is described in Example 2, or a mutated ABCA8 polypeptides whose complete cDNA may also be obtained according to the techniques described in Example 2, may be produced in a bacterial or insect cell expression system using the baculovirus vectors or in mammalian cells with or without the vaccinia virus vectors. All the methods are now widely described and are known to persons skilled in the art. A detailed description thereof will be found for example in Ausubel et al. (1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y). EXAMPLE 7: Determination of Polymorphisms/Mutations in ABCA8.
  • the detection of polymo ⁇ hisms or of mutations in the sequences ofthe transcripts or in the genomic sequence of ABCA8 may be carried out according to various protocols.
  • One method that may be used is direct sequencing.
  • the method consists of preparing the cDNAs and sequencing them directly.
  • the intron-exon structure ofthe transcript For subjects for whom only DNA is available, and in the case of a transcript where the structure ofthe corresponding gene is unknown or partially known, it may be beneficial to determine the intron-exon structure ofthe transcript as well as the genomic sequence ofthe corresponding gene. This involves, in a first instance, isolating the genomic DNA BAC or cosmid clone(s) corresponding to the transcript studied, sequencing the insert ofthe corresponding clone(s), and determining the intron-exon structure by comparing the cDNA sequence to that ofthe genomic DNA obtained.
  • the technique of detection of mutations by direct sequencing consists of comparing the genomic sequences of ABCA8 obtained from homozygotes for the disease or from at least 8 individuals (4 individuals affected by the pathology studied and 4 individuals not affected) or from at least 32 unrelated individuals from the studied population. Sequence divergences constitute polymo ⁇ hisms. All modifications ofthe amino acid sequence ofthe wild-type protein may be mutations capable of affecting the function of a protein, which may yield information regarding the mutation(s) ofthe disease (denoted genotype-phenotype correlation) in the pedigree, or of a pharmacological response to a therapeutic molecule in the pharmacogenomic studies.
  • EXAMPLE 8 Identification of a Causal Gene for a Disease Linked to ABCA8
  • RNA specific to affected or non-affected individuals makes it possible to detect notable variations in the level of expression ofthe gene studied, in particular the absence of transcription of the gene.
  • Polyclonal antibodies specific for a human ABCA8 polypeptide may be prepared as described herein in rabbits and chicks by injecting a synthetic polypeptide fragment derived from an ABCA8 protein, including all or part of an amino acid sequence as shown in SEQ ID NO: 2, for instance a fragment chosen from within residues 597-636 of SEQ ID NO: 2. These polyclonal antibodies are used to detect and/or quantify the expression ofthe ABCA8 gene in cells and animal models by immunoblotting and/or immunodetection.
  • a biological activity of ABCA8 may be monitored by quantifying the cholesterol fluxes induced by apoA-I using cells transfected with the vector pCMV-ABCI which have been loaded with cholesterol (Remaley et al, 1997, ATVB, 17:1813).
  • EXAMPLE 10 Expression in vivo of the ABCA8 Gene in Various Animal Models:
  • An appropriate volume (e.g., 100 to 300 ⁇ l) of a medium containing the purified recombinant adenovirus (pABCA-AdV or pLucif-AdV) containing from 10 8 to 10 9 lysis plaque- forming units (pfu) are infused into the Saphenous vein of mice (C57BL/6, both control mice and models of transgenic or knock-out mice) on day 0 ofthe experiment.
  • the evaluation ofthe physiological role ofthe ABCA8 protein in the transport of cholesterol or inflammatory lipid substances is carried out by determining the total quantity of cholesterol or appropriate inflammatory lipid substances before (day zero) and after (days 2, 4, 7, 10, 14) the administration ofthe adenovirus.
  • transgenic mice and rabbits over-expressing gene may be produced, in accordance with the teaching of Vaisman (J. Biol. Chem. 270(20): 12269-12275, 1995) and Hoeg (Proc. Nat. Acad. Sci. USA. 93(21):11448-11453, 1996) using constructs containing the human ABCA8 cDNA under the control of endogenous promoters such as ABCA8 or CMV or apoE.
  • An appropriate volume e.g., 100 to 300 ⁇ l
  • a medium containing the purified recombinant adenovirus (pABCA-AdV or pLucif-AdV) containing from 10 8 to 10 9 lysis plaque- forming units (pfu) are infused into the Saphenous vein of mice (C57BL/6, both control mice and models of transgenic or knock-out mice) on day 0 ofthe experiment.
  • the evaluation ofthe physiological role ofthe ABCA8 protein in the transport of cholesterol or inflammatory lipid substances is carried out by determining the total quantity of cholesterol or appropriate inflammatory lipid substances before (day zero) and after (days 2, 4, 7, 10, 14) the administration ofthe adenovirus.
  • transgenic mice and rabbits over-expressing gene may be produced, in accordance with the teaching of Vaisman (J. Biol. Chem. 270(20): 12269-12275, 1995) and Hoeg (Proc. Nat. Acad. Sci. USA. 93(21):11448-1 1453 1996) using constructs containing the human ABCA8 cDNA under the control of endogenous promoters such as ABCA8 or CMV or apoE.
  • endogenous promoters such as ABCA8 or CMV or apoE.
  • the evaluation ofthe long-term effect ofthe expression of ABCA8 on the kinetics ofthe lipids involved in the mediation ofthe inflammation may be carried out as described above.
  • EXAMPLE 11 Sequence Analysis of Human ABCA8 cDNA.
  • EXAMPLE 12 Phylogenetic analysis of the relationship between the ABCA8 to other ABCA transporters.
  • This example provides a description of how the evolutionary relationship between human ABCA8 and other ABCA transporters was determined.
  • Phylogenetic analysis was performed using the ATP-binding domain, which is conserved across the species and in different subfamilies without large deletions or insertions (see Bodenmiller et al, DNA Seq. 13(2): 77-83, 2002). Analysis was conducted using both ATP-binding domains, as all twelve human ABCA subfamily members known to date contain two such domains.
  • ABCA8 belongs to the subgroup inside the A subfamily, which contains the best-characterized members of this subfamily, ABCAl (see Tanaka et al,
  • the second subgroup includes five genes that form a tandem cluster at chromosome 17q24.
  • ABCA8 has a short (about 90 amino acids) region of similarity at the N-terminus, and larger region of similarity starting from amino acid 3070. This suggests that the large size of ABCA8 is due to the large insertion between these positions at the first extracellular domain.
  • Embodiments of this disclosure provide several ABCA8 proteins and nucleic acid molecules, and methods of isolating, making, and using these molecules. Further embodiments provide methods for ameliorating, treating, detecting, prognosing and diagnosing diseases related to expression of ABCA8. It will be apparent that the precise details ofthe methods described may be varied or modified without departing from the spirit ofthe described invention. We claim all such modifications and variations that fall within the scope and spirit ofthe claims below.

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Abstract

La présente invention concerne des acides nucléiques correspondant au gène et à l'ADNc ABCA8, la protéine ABCA8, ainsi que des méthodes de production et d'utilisation de ces molécules. Cette invention concerne également des méthodes visant à améliorer, traiter, détecter, pronostiquer et diagnostiquer des maladies et des états que l'on croit associés à une expression modifiée de ABCA8. Parmi ces maladies et états figurent l'hypercholestérolémie, la résistance aux médicaments, la dégénérescence rétinienne ou la maladie neurologique. Cette invention concerne également des trousses et des compositions pharmaceutiques.
PCT/US2002/033496 2001-10-19 2002-10-18 Acides nucleiques et proteines abca8 et leurs utilisations Ceased WO2003064591A2 (fr)

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Publication number Priority date Publication date Assignee Title
WO2005056796A1 (fr) * 2003-12-15 2005-06-23 Noab Biodiscoveries Inc. Materiaux et methodes d'analyse de l'expression du gene transporteur de la cassette se liant a atp

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* Cited by examiner, † Cited by third party
Title
NAGASE ET AL.: 'Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro' DNA RESEARCH vol. 5, 1998, pages 355 - 364, XP002924150 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005056796A1 (fr) * 2003-12-15 2005-06-23 Noab Biodiscoveries Inc. Materiaux et methodes d'analyse de l'expression du gene transporteur de la cassette se liant a atp

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