WO2001087921A2 - Gene responsable de la dystrophie maculaire dominante de stargardt - Google Patents

Gene responsable de la dystrophie maculaire dominante de stargardt Download PDF

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WO2001087921A2
WO2001087921A2 PCT/US2001/015464 US0115464W WO0187921A2 WO 2001087921 A2 WO2001087921 A2 WO 2001087921A2 US 0115464 W US0115464 W US 0115464W WO 0187921 A2 WO0187921 A2 WO 0187921A2
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elf
seq
protein
stargardt
macular dystrophy
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WO2001087921A3 (fr
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Konstantin Petrukhin
Wen Li
Kang Zhang
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Johns Hopkins University
Merck and Co Inc
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Johns Hopkins University
Merck and Co Inc
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Priority to US10/276,732 priority Critical patent/US7091005B2/en
Priority to EP01935445A priority patent/EP1283897A4/fr
Priority to CA002409705A priority patent/CA2409705A1/fr
Publication of WO2001087921A2 publication Critical patent/WO2001087921A2/fr
Publication of WO2001087921A3 publication Critical patent/WO2001087921A3/fr
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Priority to US11/477,042 priority patent/US7179620B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to the gene responsible for causing Stargardt-like dominant macular dystrophy, and to assays which use this gene or the protein encoded by it, and to methods of treating this condition by administering the protein.
  • Macular dystrophy is a term applied to a heterogeneous group of diseases that collectively are the cause of severe visual loss in a large number of people.
  • a common characteristic of macular dystrophy is a progressive loss of central vision resulting from the degeneration of photoreceptor cells in the retinal macula.
  • the end stage of the disease results in legal blindness.
  • macular dystrophy More than 20 types of macular dystrophy are known: e.g., age-related macular dystrophy, Stargardt-like dominant macular dystrophy, recessive Stargardt's disease, atypical vitelliform macular dystrophy (VMDl), Usher Syndrome Type IB, autosomal dominant neovascular inflammatory vitreoretinopathy, familial exudative vitreoretinopathy, and Best's macular dystrophy (also known as hereditary macular dystrophy or Best's vitelliform macular dystrophy (VMD2).
  • VMD2 hereditary macular dystrophy
  • Best's vitelliform macular dystrophy See Sullivan & Daiger, 1996, Mol. Med. Today 2:380-386.
  • Stargardt-like dominant macular dystrophy (also called autosomal dominant macular atrophy) is a juvenile-onset macular degeneration. Patients afflicted with this disease generally have normal vision as young children, but during childhood, visual loss begins, which rapidly progresses to legal blindness. Clinically it is characterized by the presence of an atrophic macular lesion with sharp borders and is often associated with yellow fundus flecks.
  • the pathological features seen in Stargardt-like dominant macular dystrophy are in many ways similar to the features seen in age-related macular dystrophy (AMD), the leading cause of blindness in older patients in the developed world.
  • AMD age-related macular dystrophy
  • AMD is an extraordinarily difficult disease to study genetically, since by the time patients are diagnosed, their parents are usually no longer living and their children are still asymptomatic. Thus, family studies which have led fo the discovery of the genetic basis of many other diseases have not been practical for age-related macular dystrophy. As there are currently no widely effective treatments for AMD, it is hoped that study of Stargardt-like dominant macular dystrophy, and in particular the discovery of the underlying genetic cause of Stargardt-like dominant macular dystrophy, will shed light on age-related macular dystrophy as well. A significant proportion of the AMD cases is caused by recessive mutations in the recessive Stargardt disease gene. (Allikmets, et al 1997 Science 277:1805-1807).
  • DHA decosahexaenoic fatty acid
  • the mutant version of this gene contains a 5-base pair deletion which causes a frameshift mutation.
  • the resultant mutated protein does not function in the DHA pathway, resulting in retinal dysfunction.
  • one aspect of this invention is a nucleic acid encoding the normal form of ELF protein, which is free from associated nucleic acids.
  • the nucleic acid sequence is a DNA, and in more preferred embodiments it is a cDNA.
  • Another aspect of this invention is a nucleic acid encoding a mutant form of ELF, which is free from associated nucleic acids.
  • the nucleic acid is a DNA, and in more preferred embodiments, it is a cDNA.
  • Another aspect of this invention are the novel proteins, normal ELF and its mutant form, free from associated proteins. Also part of this invention are fragments of these proteins which retain at least one biological activity.
  • a further aspect for this invention is a method of treating individuals who suffer from Stargardt-like macular dystrophy comprising administering to the individual an effective amount of ELF protein.
  • the ELF protein may be in a pharmaceutically acceptable carrier, and it may be administered in the form of eyedrops or other ophthalmic preparation.
  • Another aspect of this invention is a method of treating individuals who suffer from Stargardt-like macular dystrophy comprising introducing a nucleic acid encoding the ELF protein into the individual.
  • This gene therapy approach may involve the use of viral vectors, such as adenovirus, or it may involve the use of plasmid DNA.
  • Yet another aspect of this invention are assays to identify if an individual is at risk for Stargardt-like macular dystrophy comprising determining if the individual's DNA contains a gene for a mutant form of ELF.
  • Another aspect of this invention is the use of ELF gene's 5' regulatory region for targeting the expression of genes specifically to photoreceptor cells of the retina for gene therapy of macular degeneration.
  • Another aspect of this invention is the use of mouse ELF DNA or mouse ELF protein corresponding to the normal or mutant form of human ELF for generating an animal model (knock-out or transgenic) that can be used for testing the anti-AMD compounds.
  • a further aspect of this invention are methods of producing long chain fatty acids using DNA encoding ELF or using ELF protein.
  • FIGURE 1 is the protein for normal human ELF protein (SEQ.ID.NO. 1).
  • the underlined amino acids represent 5 putative transmembrane segments.
  • the HXXHH motif between predicted membrane spanning regions 2 and 3 that is characteristic of dioxy iron cluster proteins is double underlined.
  • the protein fragment deleted in patients with Stargardt-like macular dystrophy is shown in italics.
  • the cytosolic carboxy-terminal dilysine motif responsible for the retrieval of trans- membrane proteins from ds-Golgi to the endoplasmic reticulum is shown in bold italics.
  • FIGURE 2 is the protein for mutant human ELF protein which causes Stargardt-like dominant macular dystrophy (SEQ.ID.NO. 2).
  • Underlined amino acids represent four of five putative transmembrane segments; the fragment of the fifth transmembrane segment that is common for normal and mutant alleles of the protein is highlighted by the dotted line.
  • the HXXHH motif between predicted membrane spanning regions 2 and 3 that is characteristic of dioxy iron cluster proteins is double underlined.
  • the protein fragment generated by the 5-bp deletion in patients with Stargardt-like macular dystrophy is shown in italics.
  • FIGURE 3 is normal human ELF cDNA (SEQ.ID.NO. 3) and the amino acid sequence (SEQ.ID.NO.1) of the human ELF protein.
  • Underlined nucleotides in bold encompassing base pairs 797- 801 represent the deletion found in patients with dominant Stargardt-like macular dystrophy.
  • the protein fragment deleted in patients with Stargardt-like macular dystrophy is shown in bold underline
  • FIGURE 4 is mutant human ELF cDNA (SEQ.ID.NO. 4) and the amino acid sequence (SEQ.ID.NO. 2) of the human mutant ELF protein.
  • the region of the protein encompassing amino acids 264-271 (bold underlined) represent a fragment generated as a result of the 5-base pair deletion in patients with dominant Stargardt-like macular dystrophy.
  • FIGURE 5 is the protein for normal mouse ELF protein (SEQ.ID.NO. 5). Underlined amino acids represent 5 putative transmembrane segments. The HXXHH motif between predicted membrane spanning regions 2 and 3 that is characteristic of dioxy iron cluster proteins is double underlined. The protein fragment similar to the human ELF fragment deleted in patients with Stargardt-like macular dystrophy is shown in italics. Cytosolic carboxy-terminal dilysine motif responsible for the retrieval of transmembrane proteins from c-s-Golgi to the endoplasmic reticulum is shown in bold italics.
  • HGURE 6 is mouse cDNA for ELF (SEQ.ID.NOS. 6) and the amino acid sequence (SEQ.ID.NO.:5) of the mouse ELF protein.
  • FIGURE 7 shows the genomic DNA sequence of the ELF gene (SEQ.ID.NO.:8). Underlined nucleotides in capitals represent exons. Initiating ATG codon in exon 1 and terminating TAA codon in exon 6 are shown in bold italics. The exact lengths of the gaps between the exons are unknown; these gaps are presented as runs of ten bold n as a convenience only.
  • FIGURE 8 shows the pairwise comparison of human and mouse ELF proteins.
  • the upper amino acid sequence shown is the human ELF protein (SEQ.ID.NO. 1).
  • the lower amino acid sequence shown is the mouse ELF protein (SEQ.ID.NO. 5).
  • the two proteins are highly identical which indicates they are true orthologues. Both proteins share the cytosolic carboxy-terminal dilysine motif responsible for the retrieval of transmembrane proteins from cis-Golgi to the endoplasmic reticulum (two lysines are located at -3 and -5 positions with respect to the carboxyl terminus).
  • FIGURE 9 depicts the sequence alignment of the human ELF protein (SEQ.ID.NO. 1) and its two yeast homologues, Elo2p (SEQ.ID.NO. 8) and Elo3p (SEQ.ID.NO. 9) from Oh et al.,1997 J. Biol. Chem 272:17376-17384.
  • the degree of homology is high enough to assign the function of elongation of fatty acids to the human ELF protein.
  • FIGURE 10 depicts the enzymatic conversions involved in the linoleic acid (n-3) and alpha-linolenic acid (n-6) pathways of essential fatty acid synthesis, including three elongation steps required of the biosynthesis of DHA
  • FIGURE 11 shows a Kyte-Doolittle hydropathy plot of human ELF. Numbers 1 to 5 mark putative transmembrane segments.
  • the hydropathy plot and membrane topology of human ELF (SEQ.ID.NO. l)are similar to those proposed for its two yeast homologues, Elo2p (SEQ.ID.NO. 8) and Elo3p (SEQ.ID.NO. 9), experimentally shown to be involved in elongation of fatty acids.
  • FIGURE 12 shows association (segregation) of the 5 base pair deletion within the ELF gene with the disease phenotype in the family with dominant
  • Stargardt-like macular dystrophy The figure shows the structure of this pedigree and four sequencing runs (boxed) of PCR fragments that represent exon 6 and adjacent intronic regions of the human ELF gene (SEQ._D.NOS.:10, 11, 12, and 13). From left to right, the runs are from A40 (father, unaffected with Stargardt-like dominant macular dystrophy), A4 (mother, affected with Stargardt-like dominant macular dystrophy), A430 (son of A4 and A40, unaffected with Stargardt-like dominant macular dystrophy), A43 (daughter of A4 and A40, affected with Stargardt-like dominant macular dystrophy). Reading the boxed chromatograms from left to right, the 5-base pair deletion shows up as appearance of double peaks starting from position 7 in the case of patients A4 and A43.
  • FIGURES 13 A and 13B show the result of in situ hybridization of the human ELF mRNA in rhesus monkey retina.
  • FIGURE 13 A shows specific expression in the inner segments of photoreceptor cells with the antisense probe.
  • FIGURE 13B shows the hybridization with the sense control probe (sense probe is not complementary to the ELF mRNA).
  • Retinal layers are marked as RPE, retinal pigment epithelium; OS, outer segments of photoreceptors; IS, inner segments of photoreceptors; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
  • PhumGLl/HR2 is a hybridization probe that represents a fragment of the human normal ELF cDNA (SEQ.ID.NO. 4). with coordinates 561-771.
  • FIGURE 14 shows the expression pattern of the ELF gene in 10 human tissue plus retinal pigment epithelium-derived cell line ARPE19, as determined by
  • FIGURE 15 shows the expression pattern of the human ELF gene in human tissues as determined by Northern blot hybridization. The expression is prominent in the human retina; the hybridization signal is also seen in the human brain. ELF mRNA exists in two different species, similar to what was reported for its only mammal relative, the Cig30 gene (Tvrdik et al 1999, J. Biol. Chem. 274:26387-
  • FIGURE 16 shows the 5 -regulatory region of human ELF gene.
  • the initiating ATG codon in the first exon is shown in bold.
  • Sequence elements that are common to mammalian RNA polymerase II promoters CAAT box at position 1657 and four GC boxes at positions 1446, 1513, 1585, and 1744) are shown in bold and underlined.
  • Free from associated nucleic acids means the nucleic acid is not covalently linked to a nucleic acid which is naturally linked to in an individual's chromosome.
  • Free from associated proteins means the ELF protein is not located in its native cell membrane; or in the case of the mutant allele, the mutant ELF is not located in the retinal cytoplasm where it normally is found.
  • This invention relates to the identification and characterization of the mutant allele responsible for Stargardt-like macular dystrophy.
  • the designation of the gene is EFL (for Elongation of Fatty Acids).
  • EFAs Essential fatty acids
  • the most important dietary EFAs are linoleic acid and alpha-linolenic acid (ALA). These two EFAs undergo a number of biosynthetic reactions that convert them into various other EFAs.
  • FIGURE 10 depicts the biosynthetic reactions involving the two groups of EFAs, the n-6 EFAs (linoleic acid derivatives) and the n-3 EFAs (ALA derivatives).
  • EFAs are formed from linoleic acid and ALA by a series of alternating reactions involving the removal of two hydrogens coupled with the insertion of an additional double bond (desaturation) and the lengthening of the fatty acid chain by the addition of two carbons (chain elongation).
  • the end product of the ALA pathway is docosahexaenoic acid (DHA).
  • Decosahexaenoic fatty acid is a highly polyunsaturated, long- chain fatty acid, which has six double bonds and is 22 carbons in length [indicated as 22:6 (n-3), where the first number indicates chain length, the second number indicates the number of double bonds, and "n-3" indicates the position of the first double bond as its relates to the terminal methyl group].
  • DHA is a critical component of membranes in vertebrate retina, comprising up to 50% of all fatty acids in photoreceptor cells. While not wishing to be bound by theory, it appears the normal allele of ELF is involved in one of the elongation steps during DHA synthesis.
  • ELF protein is responsible for the biosynthesis of DHA, as it requires the elongation up to 24 carbon atoms with subsequent chain shortening (beta- peroxidation) to 22 carbon atoms.
  • the mutant (i.e. disease-causing allele) of ELF contains a 5 bp deletion starting at bp 797. This results in a frameshift mutation from this position through the remainder of the C-terminus. The mutation removes the C-terminal region of the ELF protein which is reasonably conserved between human and mouse (see FIGURE 8). Evolutionary conservation indicates functional significance of the protein region removed as a result of the frameshift mutation.
  • the frameshift mutation removes the targeting signal in the C-terminus which is the same sequence as those known to be responsible for targeting proteins to the endoplasmic reticulum (Gaynor et al.1994 J. Cell Biol. 127:653-665 and Schroder et al.1995 J. Cell Biol.131:895-912, both of which are incorporated by reference).
  • deficiencies in the biosynthesis of DHA or other retina-specific fatty acids with very long chain resulting from mutations in ELF would predictably lead to retinal dysfunction.
  • nucleic acids which encode either the normal allele or the mutant allele of ELF; these nucleic acids may be free from associated nucleic acids.
  • the source of the nucleic acids is a human; although this invention includes other mammalian forms, such as mouse, rat, pig, monkey and rabbit.
  • Genes encoding ELF from a non-human mammal can be obtained by using the human DNA as a probe in libraries of the retina using standard biotechnological techniques, and one aspect of this invention is a method of isolating a non-human nucleic acid encoding an ELF protein comprising probing a retinal library of a non-human mammal.
  • the probe is preferably from the human or mouse DNA,
  • the term "gene” specifically refers to the protein-encoding portion of the gene, i.e. the structural gene, and specifically does not include regulatory elements such as promoters, enhancers, transcription termination regions and the like.
  • the gene may be a cDNA or it may be an isolated form of genomic DNA.
  • isolated means that the DNA is physically separated from the DNA which it is normally covalently attached to in the chromosome. This includes DNA with a heterologous promoter and DNA which has its native regulatory sequences, but is not present in its native chromosome.
  • the ELF genes of this invention may have their own regulatory sequences operatively linked, or one may, using known biotechnology techniques, operatively linked heterologous regulatory regions. Such regulatory regions are well known, and include such promoters as the CMN promoter, rod- specific promoter of the rodopsin gene, retinal pigment epithelium-specific promoters of bestrophin or RPE65 genes.
  • ELF D ⁇ A human ELF D ⁇ A
  • EBO-pSV2-neo ATCC 37593
  • pBPV- 1(8-2) ATCC 37110
  • pdBPV-MMTneo ATCC 37224
  • pRSVgpt ATCC 37199
  • pRSVneo ATCC 37198
  • pSV2-dhfr ATCC 37146
  • ELF genes regardless of species and allelic form
  • operatively linked regulatory regions an "ELF expression cassette”
  • Vectors which are preferred include plasmids and, to a lesser degree, viral vectors. The choice of vector will often be dependent upon the host cell chosen.
  • Cells which are preferred host cells include but are not limited to: ARPE-19, RPE-J, Y79, L cells L-M(TK “ ) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • a further aspect of this invention is a method of making an ELF protein (either a mutant or normal allelic form) comprising culturing a host cell comprising an ELF expression cassette, and recovering ELF protein.
  • a ELF gene may be integrated into a chromosome of the host cell, rather than being located on a vector.
  • the resultant ELF-expressing cell lines (comprising a heterologous ELF gene, whether on a vector or in a host's chromosome) make up yet another aspect of this invention.
  • allelic form of ELF protein (normal or mutant) which is free from associated proteins.
  • the protein is mammalian, and in more preferred embodiments, the protein is a human form.
  • Still another aspect of this invention is a method for treating, preventing or lessening the severity of Stargardt-like macular degeneration comprising administering the normal allelic form of ELF to an individual at risk of the disease or who manifests the symptoms of the disease.
  • the normal allelic form of ELF is preferably recombinantly produced.
  • the normal ELF can substitute for the defective ELF made by these individuals, and perform the normal transporting function.
  • the administration of the ELF protein is preferably in the form of a pharmaceutical composition comprising pharmaceutically acceptable diluents, excipients, and optionally stabilizers or preservatives.
  • a typical pharmaceutical composition comprises 0.1 to 95% protein and is administered once, twice or three times daily.
  • the pharmaceutical composition is preferably in the form of eyedrops, solutions or suspensions for subretinal and intravitreal injections, or slow release pellets.
  • Still another aspect of this invention is a method for in vitro biosynthesis of fatty acids with a very long chain, for example DHA.
  • Biosynthesis of DHA involves several elongation and desaturation steps (see FIGURE 10).
  • CYB5RP retina-specific delta 6 desaturase
  • CYB5RP is homologous to a delta 6 desaturase from Borago oficinalis.
  • Both CYB5RP and this Borago delta 6 desaturase unlike desaturases from higher plants, are unusual in containing a cytochrome b5-like domain fused to their N-termini (Sayanova et al., 1997, Proc. Natl. Acad. Sci. USA 94:4211-4216; hereinafter "Sayanova", which is hereby incorporated by reference).
  • the Borago desaturase has been expressed in transgenic tobacco, resulting in high levels of delta 6 desaturated fatty acids in the transgenic tobacco leaves, including high levels of ⁇ -linolenic acid (GLA) (Sayanova).
  • GLA ⁇ -linolenic acid
  • CYB5RP expressed in transgenic plants (e.g., tobacco) is expected to provide a valuable source of GLA.
  • Co-expression of the ELF cDNA in the same plant would predictably couple elongation and desaturation steps required for the production of DHA.
  • CYB5RP and ELF DNA co-expressed in transgenic plants, is expected to provide a valuable source of the important nutrient- docosahexaenoeic acid (DHA).
  • DHA nutrient- docosahexaenoeic acid
  • Another aspect of this invention is the use of mouse ELF DNA or mouse ELF protein corresponding to the normal or mutant form of human ELF for generating an animal model (knock-out or transgenic) that can be used for testing anti- AMD compounds.
  • Oligonucleotide primers designed from the mouse cDNA sequence (SEQ.ID.NOS. 6) can be used to PCR amplify a fragment of the mouse ELF gene from the DNA of 129-strain embryonic stem cells (DNA of the 129Sv/J lambda genomic library is available from Stratagen). This genomic fragment can be used to generate a construct that will, upon electroporation into the 129-strain ES cells, generate a null mutation (targeted disruption) of the ELF gene.
  • ES clones that have undergone homologous recombination with the construct can be injected into C57BL/6 blastocysts. Injected blastocytes can be transplanted into the uterus of pseudopregnant female mice. Their progeny can be selected for the germline transmission of the disrupted ELF gene and bred with 129SVEV females. The animals with heterozygous disruption of the mouse ELF gene can be bred to homozygosity.
  • Another aspect of this invention is an assay to identify individuals who are at risk for developing the symptoms of Stargardt-like macular dystrophy.
  • the children of a person who has this disease are at risk, as the disease is inherited in a dominant-Mendelian fashion.
  • the second parent is a heterozygous afflicted patient, the children have a 50% probability of developing the disease.
  • One assay in accordance with this invention is a labeled nucleic acid probe which spans the portion of the nucleic acid just 5 'to the area where the mutant deletion occurs, and includes base pairs after the deletion, which include the frameshift mutation.
  • a probe would be of any convenient length, preferably about 15 to 35 bp in length, more preferably at least about 25-30 base pairs in length. It would include a desired number of base pairs up to 796, skip 797-801, and resume at 802.
  • the probe can be constructed so that it would hybridize to the sense strand, or alternatively so that hybridization occurs with the anti-sense strand.
  • a typical probe would thus comprise (where the superscripted numeral correspond to base pair positions according to the normal allele): C790 ⁇ 791 T792 ⁇ 793 c794 795 ⁇ 796 c802 ⁇ 803 A804 C805 A806 ⁇ 807 ⁇ 808 c809
  • the probe may contain additional 5' and or 3 -terminus base pairs which are essentially identical to those in the normal allele, so that the length of the probe is at least 15 bp long, and preferably at least 25 bp long.
  • the probe includes a detection means, such as a detectable label.
  • detection means such as a detectable label.
  • Such labels including radiolabels or fluorescent labels are well known in the art.
  • the probe would include base pairs which would hybridize to the normal allelic form of the ELF gene, but would not hybridize to the mutant form.
  • Another embodiment of this invention is a method of determining if an individual is at risk of developing Stargardt-like macular dystrophy comprising obtaining a sample of the ELF protein produced by the individual, and determining whether it is the normal or mutant form. This is preferably done by determining if an antibody specific for the normal allele of the ELF protein binds to the protein produced by the individual. In an alternate embodiment of this assay, the antibody is specific for the mutant form of ELF.
  • the antibodies of these assays may be polyclonal antibodies or monoclonal antibodies.
  • the antibodies can be raised against the C-terminal peptide which is different in normal and mutant ELF proteins.
  • the antibodies can be raised against the allele-specific synthetic C-terminal peptides that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art.
  • suitable carriers e.g., serum albumin or keyhole limpet hemocyanin
  • Methods of identifying suitable antigenic fragments of a protein are known in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. 78:3824-3828; and lameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181- 186, both of which are hereby incorporated by reference.
  • ELF protein or an antigenic fragment coupled to a suitable carrier, is injected on a periodic basis into an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice.
  • the animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected antigen.
  • the injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.
  • ELF protein or an antigenic fragment coupled to a suitable carrier, is injected into an appropriate non- human host animal as above for the production of polyclonal antibodies.
  • the animal is generally a mouse.
  • the animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler & Milstein, 1975, Nature 256:495-497.
  • Antibodies A Laboratory Manual, Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.
  • Normal and mutant ELF proteins differ in size (normal ELF is 41 amino acid longer which translates in the 4 kiloDalton difference on the SDS-PAGE). Such a difference can be easily detected, so antibodies against the common parts of the two proteins can be used on Western blots to detect the presence of the mutant ELF.
  • Gene therapy may be used to introduce ELF polypeptides into the cells of target organs, e.g., the photoreceptor cells, pigmented epithelium of the retina or other parts of the retina.
  • Nucleotides encoding ELF polypeptides can be ligated into viral vectors which mediate transfer of the nucleotides by infection of recipient cells.
  • Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, and polio virus based vectors.
  • nucleotides encoding ELF polypeptides can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted transfer using ligand-nucleotide conjugates, lipofection, membrane fusion, or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Gene therapy with ELF polypeptides will be particularly useful for the treatment of diseases where it is beneficial to elevate ELF activity.
  • Promoter/5-regulatory region of the ELF gene can be used in suitable viral and non- viral vectors to target the expression of other genes specifically in the photoreceptor cells of the human retina, due to the unique photoreceptor cell specificity of the ELF gene transcription.
  • FIGURE 16 shows the promoter for human ELF.
  • DNA sequence from D6S460 was compared with GenBank database entries using the BLASTN algorithm. This comparison revealed that D6S460 is contained within the DNA sequence of PAC dJ75K24 (GenBank accession number AL035700).
  • Cycling conditions were as follows: 1) 94°C for 10 min; 2) 94°C for 30 sec; 3) 72°C for 2 min (decrease this temperature by 1.1°C per cycle); 4) 72°C for 2 min; 5)Go to step 2 fifteen more times; 6) 94°C for 30 sec; 7) 55°C for 2 min; 8) 72°C for 2 min; 9) Go to step 6 twenty four more times; 10). 72°C for 7 min; and 11) 4°C.
  • the PCR product was electrophoresed on a 2% agarose gel and DNA band was excised, purified and subjected to sequence analysis with the same primers that were used for PCR amplification.
  • DNA sequence analysis was performed using the ABI PRISMTM dy e terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase, FS (Perkin Elmer, Norwalk, CT). Following linear amplification in a Perkin-Elmer 9600, the extension products were purified and analyzed on an ABI PRISM 377 automated sequencer (Perkin Elmer, Norwalk, CT). The assembly of the DNA sequence results of this PCR product revealed that there is an additional exon between exons 2 and 4; it was later designated exon 3.
  • the DNA sequence of exon 4 was compared with the EST database using the BlastN algorithm in an attempt to identify additional cDNA sequences.
  • This analysis identified a mouse skin EST (GenBank accession number AA791133) with very high degree of similarity to exon 4 of the human ELF gene.
  • the DNA sequence of the mouse skin EST AA791133 was compared with the genomic sequence of PAC 92c4. Despite the differences between the mouse and human sequences caused by evolutionary divergence, this analysis was able to reveal two additional human exons with PAC 94c4; there were later called exons 5 and 6. This finding defined the order of the exons in ELF cDNA as 5 - ex2-ex3-ex4-ex5-ex6-3'.
  • forward and reverse PCR primers from known exons of the ELF gene were synthesized and used to PCR amplify CG1CE cDNA fragments from human retina "Marathon-ready" cDNA (Clontech, Palo Alto, CA).
  • forward primer from ex2 63exDLl : GTG TGG AAA ATT GGC CTC TG
  • reverse primer from ex6 63exHRl: CAT GGC TGT TTT TCC AGC TT
  • PCR reaction was performed using the Taq Gold DNA polymerase (Perkin Elmer, Norwalk, CT) in the reaction buffer supplied by the manufacturer with the addition of dNTPs, primers, and approximately 0.5 ng of human retina cDNA.
  • PCR products were electrophoresed on a 2% agarose gel and DNA bands were excised, purified and subjected to sequence analysis with the same primers that were used for PCR amplification.
  • the assembly of the DNA sequence results of these PCR products confirmed the cDNA sequence assembled from ELF exons and corrected the sequencing errors present in the database entry for PAC 92c4.
  • RACE is an established protocol for the analysis of cDNA ends. This procedure was performed using the Marathon RACE template from human retina, purchased from Clontech (Palo Alto, CA). cDNA primer from exon 2 (63exDRl: AGG TTA AGC AAA ACC ATC CCA) (SEQ.ID.NO. 20) in combination with a cDNA adaptor primer API (CCA TCC TAA TAG GAC TCA CTA TAG GGC ) (SEQ.ID.NO.:21) were used in 5' RACE.
  • a nested PCR reaction was performed using nested adaptor primer AP2 (ACT CAC TAT AGG GCT CGA GCG GC) (SEQ.ID.NO.:22) and gene specific primer 63exDR2 (AGG TTC TCG GTC CTT CAT CC) (SEQ.ID.NO.:23).
  • the PCR product was separated from the unincorporated dNTP's and primers using Qiagen, QIAquick PCR purification spin columns using standard protocols and resuspended in 30 ⁇ l of water. The products were analyzed on ABI 377 sequencers according to standard protocols.
  • the PCR fragment obtained in the 5 'RACE reaction was assembled into a contig with the ELF cDNA fragment covering exons 2 to 6; the DNA sequence of the resulted cDNA encodes a full-length ELF protein; the order of the exons in ELF cDNA was defined as 5'- exl-ex2-ex3-ex4-ex5-ex6-3'
  • EXAMPLE 2 Stargardt-like dominant macular dystrophy is associated with the 5-bp deletion in the evolutionary conserved region of the ELF gene
  • Genomic DNA from the patients and control individuals from three pedigrees having dominant Stargardt-like macular dystrophy (families A, C, and D) was amplified by PCR using the following primer pair: 63exH_Left (GAA GAT GCC GAT GTT GTT AAA AG) (SEQ.ID.NO.:24) 63exH_Right (GTC AAC AAC AGT TAA GGC CCA) (SEQ.ID.NO. 19)
  • This primer pair amplifies a genomic fragment that contains exon 6 and an adjacent intronic region.
  • PCR products produced using the primer sets mentioned above were amplified in 50 ⁇ l reactions consisting of Perkin-Elmer 10 x PCR Buffer, 200 mM dNTP's, 0.5 ul of Taq Gold (Perkin-Elmer Corp., Foster City, CA), 50 ng of patient DNA and 0.2 ⁇ M of forward and reverse primers. Cycling conditions were as follows: 1) 94°C for 10 min; 2) 94°C for 30 sec; 3) 72°C for 2 min (decrease this temperature by 1.1 °C per cycle); 4) 72 °C for 2 min; 5) Go to step 2 fifteen more times; 6) 94°C for 30 sec. 7) 55°C for 2 min; 8) 72°C for 2 min; 9) Go to step 6 twenty four more times; 10) 72°C for 7 min; and 11) 4°C.
  • FIGURE 12 The results of this experiment in four individuals from family A is shown in FIGURE 12.
  • the figure shows a small branch of this pedigree and four sequencing runs (boxed) of PCR fragments that represent exon 6 and adjacent intronic regions of the human ELF gene. From left to right, the runs are from A40 (father, unaffected with Stargardt-like dominant macular dystrophy), A4 (mother, affected with Stargardt-like dominant macular dystrophy), A430 (son of A4 and A40, unaffected with Stargardt-like dominant macular dystrophy), A43 (daughter of A4 and A40, affected with Stargardt-like dominant macular dystrophy).
  • EXAMPLE 3 Expression studies of the ELF gene RT-PCR RT-PCR experiments were performed on "quick-clone" human cDNA samples available from Clontech, Palo Alto, CA.
  • ARPE-19 cDNA was prepared according to standard protocols. cDNA samples from heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, retina, testis, and human retinal pigment epithelium cell line ARPE-19 were amplified with primers 63exGLl (CCC AGT TGA ATT CCT TTA TCC A) (SEQ.ID.NO. 18) and 63exHRl (CAT GGC TGT TTT TCC AGC TT) (SEQ.ID.NO.
  • the ELF gene was found to be expressed in human retina only (HGURE. 14).
  • Northern blots containing poly(A+)-RNA from different human tissues were purchased from Clontech, Palo Alto, CA. The blot contained human heart, brain placenta, lung, liver, skeletal muscle, kidney, and pancreas poly(A+)-RNA.
  • a custom-made blot containing human retina, brain, and ARPE-19 poly(A + )-RNA was ordered from FRP Grating. Primers 63exDLl (GTG TGG AAA ATT GGC CTC TG) (SEQ.ID.NO.
  • the probe hybridized to an mRNA transcript that is uniquely expressed in the human retina (see Figure 15). Weaker hybridization signal is also seen in the human brain. ELF mRNA exists in two different species, similar to what was reported for its only mammal relative, the Cig30 gene (Tvrdik et al., J. Biol. Chem., 1999, 274:26387-26392; which is hereby incorporated by reference).
  • SEQ.ID.NO. 18 and 63exHRl (CAT GGC TGT TTT TCC AGC TT) (SEQ.ID.NO. 17) were used to amplify a PCR product from the "quick-clone" human retina cDNA available from Clontech, Palo Alto, CA. This product was subcloned into the pCR- Script vector (Stratagene) giving the plasmid called phumGLl/HR2. This plasmid served as a hybridization probe and represented a fragment of the human normal ELF cDNA with coordinates 561-771. In situ hybridization was carried out on sections of rhesus monkey retina according to standard protocols.
  • RPE retinal pigment epithelium
  • OS outer segments of photoreceptors
  • IS inner segments of photoreceptors
  • ONL outer nuclear layer
  • OPL outer plexiform layer
  • INL inner nuclear layer
  • J-PL inner plexiform layer
  • GCL ganglion cell layer.

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Abstract

On a identifié le gène responsable de la dystrophie maculaire de Stargardt, ainsi que sa forme allèle normale. Ce gène mutant code une protéine mutante contenant un décalage de cadre de lecture, ce qui a pour effet une synthèse anormale des acides gras et leur transport dans la rétine. L'invention concerne également des essais concernant la dystrophie maculaire de Stargardt et des thérapies.
PCT/US2001/015464 2000-05-16 2001-05-11 Gene responsable de la dystrophie maculaire dominante de stargardt Ceased WO2001087921A2 (fr)

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CA002409705A CA2409705A1 (fr) 2000-05-16 2001-05-11 Gene responsable de la dystrophie maculaire dominante de stargardt
US11/477,042 US7179620B2 (en) 2000-05-16 2006-06-28 Gene responsible for stargardt-like dominant macular dystrophy

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Cited By (8)

* Cited by examiner, † Cited by third party
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WO2002044320A3 (fr) * 2000-11-29 2003-01-09 Xenon Genetics Inc Genes humains de l'elongase, leurs utilisations et composes destines a leur modulation
WO2010011944A2 (fr) 2008-07-25 2010-01-28 Wagner Richard W Procédés de criblage de protéines
US9333202B2 (en) 2012-05-01 2016-05-10 The Trustees Of Columbia University In The City Of New York Non-retinoid antagonists for treatment of age-related macular degeneration and stargardt disease
US9434727B2 (en) 2014-04-30 2016-09-06 The Trustees Of Columbia University In The City Of New York Substituted 4-phenylpiperidines, their preparation and use
US9637450B2 (en) 2013-03-14 2017-05-02 The Trustees Of Columbia University In The City Of New York Octahydrocyclopentapyrroles, their preparation and use
US9938291B2 (en) 2013-03-14 2018-04-10 The Trustess Of Columbia University In The City Of New York N-alkyl-2-phenoxyethanamines, their preparation and use
US9944644B2 (en) 2013-03-14 2018-04-17 The Trustees Of Columbia University In The City Of New York Octahydropyrrolopyrroles their preparation and use
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Publication number Priority date Publication date Assignee Title
WO2000070945A2 (fr) * 1999-05-20 2000-11-30 Karolinska Innovations Ab Genes impliques dans l'allongement des chaines d'acides gras et utilisations associees

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002044320A3 (fr) * 2000-11-29 2003-01-09 Xenon Genetics Inc Genes humains de l'elongase, leurs utilisations et composes destines a leur modulation
US7297523B2 (en) 2000-11-29 2007-11-20 Xenon Pharmaceuticals Inc. Human elongase genes and uses thereof
WO2010011944A2 (fr) 2008-07-25 2010-01-28 Wagner Richard W Procédés de criblage de protéines
US9333202B2 (en) 2012-05-01 2016-05-10 The Trustees Of Columbia University In The City Of New York Non-retinoid antagonists for treatment of age-related macular degeneration and stargardt disease
US10787453B2 (en) 2013-03-14 2020-09-29 The Trustees Of Columbia University In The City Of New York Octahydropyrrolopyrroles their preparation and use
US10421720B2 (en) 2013-03-14 2019-09-24 The Trustees Of Columbia University In The City Of New York Octahydrocyclopentapyrroles, their preparation and use
US12473302B2 (en) 2013-03-14 2025-11-18 The Trustees Of Columbia University In The City Of New York 4-phenylpiperidines, their preparation and use
US9926271B2 (en) 2013-03-14 2018-03-27 The Trustees Of Columbia University In The City Of New York Octahydrocyclopentapyrroles, their preparation and use
US9938291B2 (en) 2013-03-14 2018-04-10 The Trustess Of Columbia University In The City Of New York N-alkyl-2-phenoxyethanamines, their preparation and use
US9944644B2 (en) 2013-03-14 2018-04-17 The Trustees Of Columbia University In The City Of New York Octahydropyrrolopyrroles their preparation and use
US11919913B2 (en) 2013-03-14 2024-03-05 The Trustees Of Columbia University In The City Of New York 4-phenylpiperidines, their preparation and use
US10273243B2 (en) 2013-03-14 2019-04-30 The Trustees Of Columbia University In The City Of New York 4-phenylpiperidines, their preparation and use
US11028098B2 (en) 2013-03-14 2021-06-08 The Trustees Of Columbia University In The City Of New York 4-phenylpiperidines, their preparation and use
US9637450B2 (en) 2013-03-14 2017-05-02 The Trustees Of Columbia University In The City Of New York Octahydrocyclopentapyrroles, their preparation and use
US10570148B2 (en) 2013-03-14 2020-02-25 The Trustees Of Columbia University In The City Of New York N-alkyl-2-phenoxyethanamines, their preparation and use
US9434727B2 (en) 2014-04-30 2016-09-06 The Trustees Of Columbia University In The City Of New York Substituted 4-phenylpiperidines, their preparation and use
US10913746B2 (en) 2014-04-30 2021-02-09 The Trustees Of Columbia University In The City Of New York Substituted 4-phenylpiperidines, their preparation and use
US10407433B2 (en) 2014-04-30 2019-09-10 The Trustees Of Columbia University In The City Of New York Substituted 4-phenylpiperidines, their preparation and use
US11649240B2 (en) 2014-04-30 2023-05-16 The Trustees Of Columbia University In The City Of New York Substituted 4-phenylpiperidines, their preparation and use
US10072016B2 (en) 2014-04-30 2018-09-11 The Trustees Of Columbia University In The City Of New York Substituted 4-phenylpiperidines, their preparation and use
US12404277B2 (en) 2014-04-30 2025-09-02 The Trustees Of Columbia University In The City Of New York Substituted 4-phenylpiperidines, their preparation and use
US9777010B2 (en) 2014-04-30 2017-10-03 The Trustees Of Columbia University In The City Of New York Substituted 4-phenylpiperidines, their preparation and use

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