Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   



 

Title:  Gene responsible for stargardt-like dominant macular dystrophy
United States Patent: 
7,179,620
Issued: 
February 20, 2007

Inventors: 
Petrukhin; Konstantin (Collegeville, PA), Li; Wen (San Diego, CA), Zhang; Kang (Salt Lake City, UT)
Assignee: 
Merck & Co., Inc. (Rahway, NJ)
Appl. No.: 
11/477,042
Filed: 
June 28, 2006


 

Patheon


Abstract

The gene responsible for Stargardt-like macular dystrophy has been identified, along with its normal allelic form. The mutant gene encodes a mutant protein containing a frameshift mutation, resulting in abnormal fatty acid synthesis and transport in the retina. Also disclosed are assays for Stargardt-like macular dystrophy and therapies.

DETAILED DESCRIPITON OF THE INVENTION

In accordance with this invention, a mutant gene responsible for autosomal dominant Stargardt-like macular dystrophy has been identified and sequenced. Additionally, the normal allelic form of this gene has also been identified and sequenced.

A new gene, presently designated "ELF" (for Elongation of Fatty Acids), is potentially involved in the elongation pathway for the synthesis of decosahexaenoic fatty acid (DHA), a critical component in retinas. 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.

Thus one aspect of this invention is a nucleic acid encoding the normal form of ELF protein, which is free from associated nucleic acids. In preferred embodiments, 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. In preferred embodiments, 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.

Bioinformatic analysis revealed a weak but significant homology between ELF and a group of two yeast proteins (Elo2p and Elo3p), whose function are also the elongation of fatty acids. The Kyte-Doolittle algorithm (FIG. 11) predicts that ELF has a transmembrane organization involving five transmembrane regions which is similar to the reported transmembrane organization of Elo2p and Elo3p. The Elo2p and Elo3p proteins are necessary for the synthesis of very long chain fatty acids of up to 24 and 26 carbon atoms, respectively (Oh et al. 1997, J. Biol. Chem. 272:17376 17384, which is hereby incorporated by reference). It seems that human 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 FIG. 8). Evolutionary conservation indicates functional significance of the protein region removed as a result of the frameshift mutation. In addition, 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). This would prevent ELF protein from trafficking to the site of biosynthesis of very long chain fatty acids (membranes of the endoplasmic reticulum) Thus, 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.

There are additional observations which indicate that the genes of this invention are involved in Stargardt-like macular dystrophy. First of all, the mutant (disease-causing allelic form) has been identified in three independent families with Stargardt-like macular dystrophy. Secondly, the gene maps to the genetically defined region on human chromosome 6q14, which has been identified with Stargardt-like macular dystrophy. The ELF gene maps to the PAC clone dJ94c4 which is located in close vicinity of the genetic marker D6S460. The maximum reported lod score for D6S460 was 9.3, which is a clear indication of genetic proximity of this marker to the disease locus (Edwards et al. 1999, Am. J. Ophthal. 127:426 435.) Further, this gene was found to be exclusively expressed in the retina, specifically, in the photoreceptor cells (see FIGS. 13, 14, and 15).

Nucleic Acids

Thus, one aspect of this invention are nucleic acids which encode either the normal allele or the mutant allele of ELF; these nucleic acids may be free from associated nucleic acids. Preferably 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,

As used throughout the specification and claims, 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. As used herein, "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 (both allelic forms) 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 CMV promoter, rod-specific promoter of the rodopsin gene, retinal pigment epithelium-specific promoters of bestrophin or RPE65 genes. Commercially available mammalian expression vectors which are suitable for the expression of human ELF DNA include, but are not limited to: pMC1neo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), and pSV2-dhfr (ATCC 37146).

The ELF genes (regardless of species and allelic form) and operatively linked regulatory regions (an "ELF expression cassette) may be placed in a vector for transfer into a host cell. 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.sup.-) (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. Alternatively 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.

ELF Protein

Another aspect for this invention is an allelic form of ELF protein (normal or mutant) which is free from associated proteins. In a preferred embodiment 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 bio-synthesis of fatty acids with a very long chain, for example DHA. Biosynthesis of DHA involves several elongation and desaturation steps (see FIG. 10).

We have previously identified and patented a retina-specific delta 6 desaturase called CYB5RP (U.S. Provisional Application Ser. No. 60/103,760; PCT/US99/23253, which is hereby incorporated by reference). 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 .gamma.-linolenic acid (GLA) (Sayanova). Similarly, 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. Thus, CYB5RP and ELF DNA, co-expressed in transgenic plants, is expected to provide a valuable source of the important nutrient-docosahexaenoeic acid (DHA). The protocols for expression of foreign genes in plants are well developed and reported in the literature (Sayanova).

Animal Model

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.

The art of constructing the knock-out and transgenic mouse models is well-described in the literature and exemplified in Weng et al., 1999 Cell 98:13 23, which is hereby incorporated by reference.

Assays for Mutant Forms

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. Thus, if one parent does not have the disease, and the second parent is a heterozygous afflicted patient, the children have a 50% probability of developing the disease. As the children begin life with normal eyesight, there is time to intervene with protein therapy to reduce the severity, delay onset, or even completely prevent the symptoms from developing.

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. Referring to the normal allele (SEQ.ID.NO. 3), 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): C.sup.790 T.sup.791 T.sup.792 T.sup.793 C.sup.794 T.sup.795 T.sup.796 C.sup.802 T.sup.803 A.sup.804 C.sup.805 A.sup.806 T.sup.807 T.sup.808 C.sup.809 (SEQ.ID.NO. 14). 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. Generally the probe includes a detection means, such as a detectable label. Such labels, including radiolabels or fluorescent labels are well known in the art.

In an alternative embodiment, 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. Methods of identifying suitable anti genic fragments of a protein are known in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. 78:3824 3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181 186, both of which are hereby incorporated by reference.

For the production of polyclonal antibodies, 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.

For the production of monoclonal antibodies, 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. In the case of monoclonal 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. For a fuller description of the production of monoclonal antibodies, see 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

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. Alternatively, 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. FIG. 16 shows the promoter for human ELF.
 


Claim 1 of 8 Claims

1. An isolated Elongation of Fatty Acids (ELF) protein, free from associated proteins, comprising the amino acid sequence set forth in FIG. 3 (SEQ ID NO: 1).

 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]