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WO2002059369A2 - Compositions et procedes de traitement des maladies liees a une mauvaise regulation du cholesterol - Google Patents

Compositions et procedes de traitement des maladies liees a une mauvaise regulation du cholesterol Download PDF

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WO2002059369A2
WO2002059369A2 PCT/US2001/047962 US0147962W WO02059369A2 WO 2002059369 A2 WO2002059369 A2 WO 2002059369A2 US 0147962 W US0147962 W US 0147962W WO 02059369 A2 WO02059369 A2 WO 02059369A2
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hei
sequence
gene
disease
ofthe
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PCT/US2001/047962
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WO2002059369A3 (fr
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Peter Lobel
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University Of Medicine And Dentistry New Jersey Medical School
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of molecular biology, cholesterol storage disease, cholesterol regulation, and cardiovascular disease.
  • this invention provides the genetic basis for Niemann-Pick C2 disease and sets forth novel compositions and methods with which to treat this disease as well as other diseases associated with faulty cholesterol regulation, such as cardiovascular disease, atherosclerosis, Alzheimer's disease and diabetes. This is due to the fact that the gene responsible for Niemann-Pick C2 disease, HEI, plays a vital role in cellular cholesterol trafficking.
  • NP-A and NP-B are autosomal recessive disorders linked to a deficiency in the enzyme sphingomyelinase (ASM) (1).
  • ASM sphingomyelinase
  • NP-A or NP- B ASM does not properly metabolize sphingomyelin, leading to an accumulation of this lipid within the cell (2).
  • both NP-A and NP-B result from deficiencies in the same enzyme, they exhibit drastically different clinical results. Patients with NP-A exhibit severe neurological effects, generally leading to death by 2 to 3 years of age, while patients with NP-B generally have little or no neurological involvement, and may survive into late childhood or adulthood (2).
  • NP-C Niemann-Pick disease type C
  • NP-C1 is an autosomal recessive lipid storage disorder characterized by progressive deterioration ofthe central nervous system, visceral symptoms, and premature death (3).
  • NP-C1 is substantially more common than NP-C2, with a rate of occurrence estimated at 1 in 10 5 live births, as opposed to an occurrence of only 8 l ⁇ iown cases of NP- C2 worldwide.
  • the most prominent feature ofthe NP-C lesion is lysosomal sequestration of LDL-derived cholesterol, which results in downstream effects on cholesterol homeostasis (4, 5).
  • NP-C cells demonstrate a deficiency in both H and L ferritins, suggesting that they prevent utilization of iron for the synthesis of cytosolic ferritin (6).
  • LDL particles transport dietary cholesterol to fibroblasts, where they enter the cell via LDL receptor-mediated endocytosis. After entering the cell, vesicles containing the LDL fuse with lysosomes. The LDL particle is degraded, and free cholesterol is released into the cytosol (7). Little is known about how free cholesterol is transported from the lysosome to the endoplasmic reticulum, plasma membrane, and other
  • NP-C In patients with NP-C, cholesterol transport is faulty, which results in accumulation of LDL-derived cholesterol within the lysosome. Because of this, NP-C cells exhibit massive cholesterol storage as demonstrated by filipin staining (9, 10). In addition, NP-C cells display an increase in endogenous cholesterol synthesis, leading to further cholesterol accumulation. In a healthy cell, cholesterol released from the lysosome enters a feedback loop whereby it downregulates endogenous cholesterol synthesis by inhibiting the activity of HMG-CoA. In NP-C cells, this feedback cycle is defective because free cholesterol is not released by the lysosome. The result is increased cholesterol synthesis, leading to a further accumulation of cholesterol within the cell.
  • Somatic cell hybridization experiments using skin fibroblast cultures from unrelated NP-C patients demonstrated the existence of a major complementation group comprising ⁇ 95% of cases, designated NP-C1, and a minor complementation group, designated NP-C2 (9, 10, 11).
  • the NP-C1 gene has been identified and mapped to chromosome 18ql 1 (12).
  • the defect responsible for NP-C2 has been excluded from this region by linkage analysis (9).
  • NP-C patients from both complementation groups demonstrate similar clinical and biochemical phenotypes, suggesting that the genes responsible for each may interact or function sequentially in a common metabolic pathway.
  • NPC1 encodes an integral transmembrane protein consisting of 1278 amino acids, with a lysosomal targeting motif and a putative sterol-sensing domain (12).
  • NPC1 resembles a family of bacterial permeases that transport various substances, including fatty acids, through the bacterial cell membrane (13).
  • NPC1 has a permease-like pump function, but the implication for cholesterol transport is unclear (13).
  • NP-C2 is extremely rare, with only eight total cases having been reported worldwide (14).
  • the present invention identifies HEI as the gene responsible for NP-C2 disease.
  • HEI was originally cloned by differential screening of a human epididymal cDNA library, and found to be highly expressed in all parts ofthe human epididymis (15, 16). The complete sequence of HEI is available (AC005479). Characterization ofthe full-length HEI gene revealed that it is a single copy gene encoding a 151 amino acid glycoprotein of 25-27 kDa (17). .
  • the protein contains a 19 amino acid sequence that is highly conserved among mammalian species and which represents a major secretory component of epididymal fluid (18, 19, 20, 21).
  • HEI is present in numerous cDNA and SAGE libraries (see UniGene cluster Hs.119529, www. ncbi.nlm.nih.gov/UniGene).
  • a bovine homolog is present in milk (22) and bovine and murine messages are detected in several tissues (22, 23).
  • Northern blot analysis has revealed HEI mRNA in all human tissues examined, with highest levels in the testis, kidney, and liver, and lowest in lung and muscle (25). It has recently been reported that the porcine homolog of HEI specifically binds cholesterol (19).
  • NP-C2 Niemann-Pick disease type C2
  • HEI a previously described cholesterol binding protein isolated from epididymal fluid
  • HEI a ubiquitously expressed lysosomal protein. This finding is consistent with the presence of HEI in epididymal fluid, because epididymal fluid has an acidic milieu and is an abundant source of several other lysosomal proteins (18, 26, 27, 28).
  • a defect in HEI protein expression is responsible for the accumulation of LDL-derived cholesterol in P- C2 patients.
  • HEI is undetectable in fibroblasts from NP-C2 patients, but present at normal levels in control patients.
  • HEI levels are elevated in NP-Cl patients.
  • HEI HEI
  • NP-C2 cells in which HEI is inactive, exhibit large-scale cholesterol accumulation.
  • HEI inNP-C2 provides an immediate therapeutic and diagnostic weapon against both NP-C2 and other diseases involving faulty cholesterol regulation.
  • Potential targets for HEI -based therapies include atherosclerosis, Alzheimer's disease, diabetes, and cardiovascular disease. Each of these diseases is linked to defective cholesterol regulation, and an increase in patient cholesterol levels.
  • statins drugs designed to lower cholesterol levels
  • statins are not an ideal therapy for decreasing Alzheimer's, due to their sometimes dangerous side effects, which include liver damage and rhabdomyolosis (29).
  • atherosclerosis high blood cholesterol levels lead to the formation of plaques on artery walls, which can decrease blood flow and increase the risk of stroke.
  • the incidence of atherosclerosis is markedly increased in patients suffering from diabetes.
  • Cardiovascular disease is also marked by a substantial increase in cholesterol levels.
  • the connection between HEI and cholesterol regulation disclosed herein suggests that HEI therapy may be useful in combating these and other diseases in addition to NP-C2 disease.
  • HEI as the second gene of Niemann-Pick type C disease has led to the compositions and methods ofthe present invention.
  • the present invention provides pharmaceutical compositions containing the HEI polynucleotide polypeptide and antisense polynucleotide sequence, as well as vectors for their delivery to target cells and expression systems for producing the HEI polypeptide.
  • the present invention provides methods for both diagnosing and treating NP-C, as well as a method for treating other diseases in which cholesterol regulation is faulty.
  • a pharmaceutical composition consisting ofthe polynucleotide encoding HEI is provided.
  • the polynucleotide sequence is substantially similar, if not identical, to SEQ. ID. NO: 1, or is a sequence with at least 70% identity to SEQ. ID. NO: 1 as its antisense sequence.
  • the invention further includes a pharmaceutical composition consisting ofthe polypeptide sequence of HEI.
  • this polypeptide sequence is identical to the sequence of SEQ. ID. NO: 2, or is a sequence substantially similar to SEQ. ID. NO: 2.
  • the present invention includes a pharmaceutical composition consisting of a vector containing the HEI polynucleotide sequence that encodes the HEI polypeptide sequence that is substantially similar, if not identical, to SEQ. ID. NO: 2.
  • Another aspect ofthe invention is an expression system for producing the HEI polypeptide of SEQ. ID. NO: 2.
  • This expression system consists of an HEI therapeutic element that in one embodiment contains an expression cassette, which includes a HEI genetic element (e.g., a HEI DNA, cDNA, RNA, antisense polynucleotide sequence, polypeptide or protein) along with one or more additional elements, and a delivery vehicle such as a vector.
  • the delivery vehicle can be a plasmid, cosmid, bacteriophage, or virus, and includes all the appropriate, additional regulatory elements.
  • the present invention may be implicated not only in NP-C2, but also in other diseases involving faulty cholesterol regulation.
  • the invention relates to compositions and methods of treating both NP-C2 and other diseases linked to faulty cholesterol regulation, such as atherosclerosis.
  • Treatment of these conditions is preferably achieved by administering an HEI genetic element such as the polynucleotide sequence of SEQ ID NO: 1 or the polypeptide or protein of SEQ. ID. NO: 2.
  • Administration ofthe HEI genetic element can occur either by introducing purified polynucleotide or protein directly or more preferably by introduction of an expression system capable of producing the HEI polypeptide within the subject's cells.
  • this expression system consists of an HEI therapeutic element that includes an expression cassette containing an HEI genetic element along with one or more additional elements, and a delivery vehicle.
  • this delivery vehicle may be a vector such as a plasmid, cosmid, or virus, which may contain one or more additional elements.
  • the invention provides for a method of diagnosing NP-C2 within a subject by detecting a mutation in the HEI gene of sequence SEQ. ID. NO: 1. Discovery of such a mutation, be it a deletion, substitution, splice mutation, or insertion, indicates the presence of NP-C2 in said subject.
  • the method of detecting the mutation consists of either amplifying the HE-1 gene sequence and performing sequence analysis or hybridizing the HEI gene sequence with a labeled nucleic acid probe corresponding to the wild type HEI nucleotide sequence of SEQ. ID.
  • the invention also provides a method of applying the above sequence analysis to detect the potential of a subject to genetically transmit NP-C2.
  • This embodiment ofthe invention is based on the idea that a subject with a single allele mutation in the HEI gene may be able to transmit the disease without displaying any symptoms of NP-C2.
  • the present invention also features a method of diagnosing NP-C1 in a subject by detecting elevated expression levels ofthe HEI gene. This increased expression is preferably detected by analysis of either HEI mRNA levels or HEI protein levels within a subject.
  • FIG. 1 Analysis of human brain mannose 6-phosphorylated glycoproteins.
  • FIG. 2 Subcellular distribution of HEI homolog in rat liver.
  • A HEI distribution in differential centrifugation fractions.
  • B HEI distribution in sucrose density gradient fractions from control rats.
  • C HEI distribution in sucrose density gradient fractions from rats treated the nonionic detergent Triton WR1339.
  • FIG. 3. A. HEI and cathepsin D protein levels in control and mutant fibroblasts.
  • Lane 1-3 fibroblasts from unaffected control subjects
  • Lanes 4-5 fibroblasts from sea blue histiocyte disease subjects
  • Lanes 6-9 fibroblasts from NP-C1 disease subjects
  • Lanes 10-11 fibroblasts fromNP-C2 disease subjects.
  • B Schematic ofthe HEI gene and protein.
  • FIG. 4 Correction of cholesterol accumulation in NP-C2 fibroblasts.
  • A Cholesterol accumulation in the absence of supplement.
  • B Cholesterol accumulation in presence of 0.3% conditioned medium from a CHO cell line producing recombinant human HEI .
  • C Cholesterol accumulation in presence of 0.3% conditioned medium from untransfected CHO cells.
  • D Western blot comparison of HEI expression HE 1- transfected CHO cells (lane 2) and untransfected CHO cells (lane 1).
  • E Fluorescence measurements of cells from A, B, and C.
  • NP-C Niemann-Pick disease type C
  • NP-C1 is an autosomal recessive lipid storage disorder characterized by progressive deterioration of the central nervous system, visceral symptoms, and premature death (3).
  • NP-C1 is substantially more common than NP-C2, with a rate of occurrence estimated at 1 in 10 5 live births, as opposed to an occurrence of only 8 known cases of NP- C2 worldwide.
  • Man6-P mannose 6-phosphate
  • MPRs Man6-P receptors
  • Purified MPR derivatives typically bind phosphorylated lysosomal proteins with subnanomolar affinity and can be used to detect and purify Man6-P glycoproteins (31, 34).
  • a two-dimensional gel map of MPR affinity-purified proteins from human brain contained a group of proteins sharing the sameNH 2 -terminal sequence, as determined by Edman degradation (longest sequence, EPVQFKDXGSVDGVIK), which are likely to represent differentially glycosylated isoforms ofthe same protein.
  • This sequence perfectly matched the processed NH -terminus of HEI (16, 17), a 151-amino acid glycoprotein containing a 19-amino acid signal that, along with homologs from numerous mammalian species, represents a major secretory component of epididymal fluid (18, 19, 20, 21).
  • Western blotting with polyclonal antibodies against recombinant HEI confirmed the identity ofthe proteins, and probing with radiolabeled MPR verified that HEI contained the Man6-P modification.
  • HEI refers generally to an HEI polypeptide that is ubiquitously expressed in the lysosome. This HEI polypeptide is absent in fibroblasts taken from patients with NP-C2 disease, but present at normal levels in cells taken from healthy individuals. HEI is a cholesterol binding protein that appears to play a central role in intracellular cholesterol transport and regulation. "HEI activity or HEI polypeptide activity” or “biological activity ofthe HEI or HEI polypeptide” refers to the metabolic or physiologic function of said
  • HEI gene refers to a polynucleotide as defined above in accordance with the present invention, which encodes an HEI polypeptide.
  • An "HEI therapeutic” refers to a therapeutically effective amount of an HEI related genetic sequence such as, but not limited to polynucleotide, polynucleotide antisense sequence, and HEI peptide, protein or protein fragment.
  • Isolated means altered “by the hand of man” from the natural state. If an
  • isolated composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically, or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides, or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified whether by natural processes, such as posttranslational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racernization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence ofthe variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • substantially similar refers to amino acid sequences having sequence variations that do not materially affect the nature ofthe protein (i.e. the structure, stability characteristics, substrate specificity, and/or biological activity ofthe protein). With regards to amino acids, “substantially similar” refers generally to conservative substitutions and/or variations in regions ofthe polypeptide not involved in determination of structure or function.
  • nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences ofthe nucleic or amino acids thus define the differences.
  • the BLAST programs (NCBI) and parameters used therein are employed, and the DNAstar system (Madison, WI) is used to align sequence fragments of genomic DNA sequences.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometime termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule, to the substantial exclusion of hybridization ofthe oligonucleotide with single- stranded nucleic acids of non-complementary sequence.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre- determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide construct with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule ofthe invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • substantially pure refers to a "preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate to the compound of interest (e.g. cliromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • expression cassette refers to a nucleotide sequence that contains at least one coding sequence along with sequence elements that direct the initiation and termination of transcription.
  • An expression cassette may include additional sequences, including but not limited to promoters, enhancers, and sequences involved in post- transcriptional or post-translational processes.
  • Expression system refers to a system for expressing a recombinant protein within a host cell.
  • an expression system consists of a vector containing a genetic element encoding the protein to be expressed.
  • This genetic element may comprise an expression cassette, which can include one or more elements for controlling expression, including: promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes.
  • the vector used in an expression system can consist of a plasmid or cosmid, a bacteriophage such as lambda phage or Ml 3 phage, or an animal virus, such as retrovirus, lentivirus, adenovirus, herpes simplex virus (HSV), cytomegalovirus (CMV), adeno-associated virus (AAV), papillomavirus, and simian virus (SV40).
  • a vector utilized in an expression system may contain a variety of elements for controlling expression, including: promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, this vector may include an origin of replication.
  • a "coding sequence” or "coding region” refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement other transcription control elements (e.g. enhancers) in an expression vector.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • promoter refers generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns.
  • a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of
  • a "vector” is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression ofthe segment.
  • selectable element refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.
  • reporter gene refers to a gene that encodes a product that is detectable by standard methods, either directly or indirectly.
  • the term "origin of replication” refers to a fixed location within a DNA sequence that serves as the startpoint for replication. DNA polymerase and other replicative factors bind at particular DNA sequences within the origin of replication. With regards to vectors, the origin of replication provides the ability for the vector to replicate autonomously, independent ofthe host chromosome.
  • nucleic acid construct is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term "transforming DNA”.
  • nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.
  • a "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) ofthe nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature.
  • the heterologous region encodes a mammalian gene
  • the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome ofthe source organism.
  • a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • DNA construct is also used to refer to a heterologous region, particularly one constructed for use in transformation of a cell.
  • a cell has been "transformed” or “transfected” or “transduced” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome ofthe cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • the term "in vivo delivery” involves the use of any gene delivery system, such as viral- and liposome-mediated transformation for the delivery and introduction of a HEI therapeutic agent to the cells of a subject, while they remain in the subject.
  • transduction is used to describe the delivery of DNA to eukaryotic cells using viral mediated delivery systems, such as adenoviral, AAV, retroviral, or plasmid delivery gene transfer methods.
  • the viral mediated delivery system is targeted specifically to the cell, wherein delivery is sought.
  • targeted delivery systems is well known and practiced in the recombinant arts.
  • a number of methods for delivering therapeutic formulations, including DNA expression constructs (as described further below), into eukaryotic cells are known to those skilled in the art.
  • the skilled artisan will be able to deliver the therapeutic agents ofthe present invention to cells in many different but effective ways.
  • the specificity of viral gene delivery may be selected to preferentially direct the HEI gene to a particular target cell by using viruses that are able to infect particular cell types.
  • viruses that are able to infect particular cell types.
  • different viral host ranges will dictate the virus chosen for transfer.
  • “In vitro" gene delivery refers to a variety of methods for introducing exogenous DNA into a cell that has been removed from its host environment.
  • transfection is used to describe the delivery and introduction of a therapeutic agent to a cell using non- viral mediated means, these methods include, e.g., calcium phosphate- or dextran sulfate-mediated transfection; electroporation; glass projectile targeting; and the like. These methods are known to those of skill in the art, with the exact compositions and execution being apparent in light ofthe present disclosure.
  • Ex vivo gene delivery refers to the procedure wherein appropriate cells are removed from an organism, transformed, transduced, or transfected in accordance with the teachings ofthe present invention, and replaced back into a host organism, for the purpose of therapeutic restoration and/or prevention.
  • Delivery of a therapeutic agent may be carried out through a variety of means, such as by using parenteral delivery methods such as intravascular and intramuscular injection, and the like. Such methods are known to those of skill in the art of drug delivery, and are further described herein in the sections regarding pharmaceutical preparations and treatment.
  • contacted when applied to a cell is used herein to describe the process by which an HEI gene, protein or antisense sequence, and/or an accessory element (such as a an antibody or cytotoxic agent), is delivered to a target cell or is placed in direct proximity with the target cell.
  • This delivery may be in vitro or in vivo and may involve the use of a recombinant vector system. Any method may be used to contact a cell with the HEI associated protein or nucleotide sequence, so long as the method results in either increased or decreased levels of functional HEI protein within the cell.
  • mice refers to such organisms as mice, rats, rabbits, goats, horse, sheep, cattle, cats, dogs and pigs. More preferably, “mammals” refers to monkeys and apes, and most preferably it refers to humans.
  • terapéuticaally effective amount describes an amount ofthe HEI polynucleotide, antisense polynucleotide, peptide, protein, or portion thereof that is effective to bring about a desired effect when administered to a subject (e.g. an increase or decrease in cell cholesterol accumulation) within the subject.
  • the present invention provides novel compositions and methods for treating Niemann-Pick type C2 (NP-C2) disease, involving the administration of an HEI gene, polynucleotide sequence, anti-sense sequence, polypeptide, protein or fragments thereof.
  • NP-C2 Niemann-Pick type C2
  • the present inventor has determined that the HEI protein is responsible for the defective egress of cholesterol from lysosomes that characterize subjects suffering fromNP-C2. It has been discovered that the HEI protein binds cholesterol in lysosomes and may be responsible for the transport of cholesterol from the lysosome to the various cellular targets there by ensuring that free LDL-derived cholesterol can escape from the lysosomal compartment.
  • the present invention concerns compositions and methods for treating various conditions related to the abnorrnal cellular accumulation of cholesterol, specifically that associated with the defective egress of cholestrol from lysosomes, namely NP-C2.
  • the invention is based firstly on the inventor's discovery that HEI mRNA and proteins were undetected in fibroblasts cells removed from subjects diagnosed with NP-C2; and that upon the administration of a therapeutic agent that includes an HEI genetic element (e.g., an HEI polynucleotide or peptide sequence), the diseased state could be alleviated.
  • a therapeutic agent that includes an HEI genetic element (e.g., an HEI polynucleotide or peptide sequence)
  • the HEI polynucleotides to be used by the present invention include isolated polynucleotides encoding HEI polypeptides, proteins and fragments, and polynucleotides closely related thereto.
  • the present invention identifies HEI as the gene responsible for NP-C2 disease.
  • the human HEI was originally cloned by differential screening of a human epididymal cDNA library, and found to be highly expressed in all parts ofthe human epididymis (15, 16).
  • the genomic structure of HEI was determined by sequence alignments between HEI cDNA (accession number Q 15668) and genomic DNA sequence (accession number AC005479).
  • the sequence ofthe human HEI gene is set out in SEQ. ID. NO: 1.
  • the HEI gene encodes a 151 amino acid glycoprotein of 25-27 kDA.
  • HEI is present in numerous cDNA and SAGE libraries (see UniGene cluster Hs.l 19529, www.ncbi.nlm.nih.gov/UniGene).
  • a BLAST search indicates that the human HEI protein shares extensive sequence homology with epididymal proteins from P. troglodyte and M. fascicularis.
  • compositions ofthe present invention involve a pharmaceutical composition that includes polynucleotides ofthe human nucleotide sequences contained in SEQ ID NO: 1 encoding an HEI polypeptide of SEQ ID NO:l.
  • Compositions ofthe present invention further include an HEI polynucleotide sequence comprising a nucleotide sequence that has at least 70% identity over its entire length to a nucleotide sequence encoding the HEI polypeptide of SEQ ID NO: 1.
  • the nucleotide sequences encoding the HEI polypeptide of SEQ ID NO:l may be identical to the polypeptide encoding sequence contained in SEQ ID NO:2, or it may be a sequence, which as a result ofthe redundancy (degeneracy) ofthe genetic code, also encodes the polypeptide of SEQ ID NO: 1.
  • polynucleotides with at least 70% identity are preferred, more preferably at least 80% even more preferably at least 90%» identity, yet more preferably at least 95% identity are highly preferred and those with at least 98-99%) are most highly preferred.
  • HEI polynucleotides are a nucleotide sequence that has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1 as to hybridize under conditions useable for amplification or for use as a probe or marker.
  • the invention also provides polynucleotides that are complementary (e.g., antisense polynucleotide sequences) to such HEI polynucleotides.
  • polypeptides which have at least 7Q%> identity, preferably at least 80%> identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97.99% identity, to the amino acid sequence of SEQ ID NO:l, over the entire length ofthe recited amino acid sequences.
  • HEI polynucleotides (including antisense sequences) ofthe present invention may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art. The availability of nucleotide sequence information, such as the cDNA having SEQ ID NO:2, enables preparation of compositions that include isolated HEI nucleic acid molecules produced by oligonucleotide synthesis. [0101] Compostions that include synthetic HEI oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices.
  • the resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • a synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.
  • HEI genes also may be isolated from appropriate biological sources using methods known in the art.
  • HEI may be isolated from genomic libraries of any mammal, specifically, human.
  • a preferred means for isolating HEI genes is PCR amplification using genomic or cDNA templates and HEI specific primers.
  • Genomic and cDNA libraries are commercially available, and can also be made by procedures well known in the art.
  • all the appropriate nucleic acid residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used.
  • the strategy of oligonucleotide design is well known in the art.
  • compositions of nucleic acids having the appropriate level of sequence homology may be identified by using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, according to the method of Sambrook et al., using a hybridization solution comprising: 1.0% SDS, up to 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 0.05%) sodium pyrophosphate (pH7.6), 5x Denhardt's solution, and 100 microgram ml denatured, sheared salmon sperm DNA.
  • Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes to 1 hour at 37°C in 2X SSC and 0.1% SDS; (4) 2 hours at 45-55°C in 2X SSC and 0.1% SDS, changing the solution every 30 minutes. [0104]
  • One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified percent identity is set forth by (Sambrook et al., 1989, supra):
  • T m 81.5°C + 16.6Log [Na+] + 0.4 1 (% G-C) - 0.63 (% formamide) - 600/#bp in duplex
  • [N+] [0.368] and 50%> formamide, with GC content of 42% and an average probe size of 200 bases, the T m is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1%> decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • the stringency ofthe hybridization and wash depend primarily on the salt concentration and temperature ofthe solutions. In general, to maximize the rate of annealing ofthe probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20 - 25 °C below the calculated T m ofthe ofthe hybrid. Wash conditions should be as stringent as possible for the degree of identity ofthe probe for the target. In general, wash conditions are selected to be approximately 12 - 20°C below the T m ofthe hybrid. In regard to the nucleic acids ofthe current invention, a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's
  • nucleic acids to be used in the present invention may be maintained as DNA in any convenient cloning vector.
  • clones are maintained in plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA), that is propagated in a suitable E. coli host cell.
  • H ⁇ 1 polynucleotide can be utilized to generate a pharmaceutical composition consisting of an effective amount of a purified H ⁇ 1 polynucleotide and an acceptable carrier.
  • this polynucleotide has at least 70% similarity with the sequence of S ⁇ Q. ID. NO: 1, and encodes a polypeptide sequence substantially similar to S ⁇ Q. ID. NO: 2.
  • H ⁇ 1 polynucleotides can comprise the genetic element that is incorporated into an expression vector. Such incorporation allows for the therapeutic expression of H ⁇ 1 in a host cell, which can ameliorate certain disease states in which cholesterol transport and regulation is faulty.
  • H ⁇ 1 polynucleotides that can be utilized in this therapeutic expression system include H ⁇ 1 DNA, cDNA, RNA, or antisense polynucleotides.
  • Antisense H ⁇ 1 polynucleotides can be utilized to downregulate H ⁇ 1, which could be of therapeutic benefit in any disease in which there is excess lysosomal cholesterol transport.
  • the present invention relates to pharmaceutical compositions that include human H ⁇ 1 polypeptides (or H ⁇ 1 proteins).
  • the human H ⁇ 1 polypeptides include the polypeptide of S ⁇ Q ID NO:l; as well as polypeptides comprising the amino acid sequence of S ⁇ Q ID NO:l; and polypeptides comprising the amino acid sequences which are substantially similar to the amino acid sequence of S ⁇ Q ID NO:l, that is they have at least 70% identity to that of S ⁇ Q ID NO:l, over its entire length.
  • H ⁇ 1 polypeptides exhibit at least one biological activity of H ⁇ 1.
  • the present invention further provides for a pharmaceutical composition that includes a polypeptide that comprises an amino acid sequence which has at least 80% identity, more preferably at least 90%> identity, yet more preferably at least 95% identity, most preferably at least 97-99%) identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • the HEI polypeptides to be used in the compositions and methods ofthe present invention, may be in the form ofthe "mature" protein or may be a part of a larger protein such as a fusion protein or part of smaller fragments ofthe HEI polypeptide that maintain functionality akin to the wild-type protein.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, ofthe amino acid sequence of the aforementioned HEI polypeptides.
  • Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of HEI polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • HEI proteins and polypeptides to be used in the compositions and methods of the present invention can be isolated, purified and prepared in any suitable manner, such as those well known in the art. For instance, the production ofthe proteins using in vitro expression is a method well known in the art.
  • a cDNA or gene may be cloned into an appropriate in vitro transcription vector, for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system.
  • in vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin, or BRL, Rockville, Maryland.
  • larger quantities of HEI encoded polypeptide may be produced by expression in a suitable prokaryotic or eukaryotic system.
  • a HEI DNA molecule such as the coding portion of SEQ ID NO:2 may be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell.
  • a HEI DNA molecule such as the coding portion of SEQ ID NO:2
  • a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell.
  • Such vectors comprise the regulatory elements necessary for expression ofthe HEI DNA in the host cell, positioned in such a manner as to permit expression ofthe DNA into the host cell.
  • Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences. Plasmids specifically designed to express and secrete foreign proteins are available from commercial sources. For example, if expression is desired in E. coli, commonly used plasmids include pTrcPPA (Pharmacia); pPROK-C and pKK233-2 (Clontech); and pNH8a, pNH16a, pcDNAII and pAX (Stratagene), among others. [0113]
  • the H ⁇ 1 proteins produced by in vitro transcription and translation or by gene expression in a recombinant procaryotic or eukaryotic system may be purified according to methods known in the art.
  • Recombinant proteins can be substantially purified by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or fusion proteins such as His tags. Such methods are commonly used by skilled practitioners.
  • the H ⁇ 1 proteins thus prepared may then be analyzed according to standard procedures. For example, the protein may be subjected to amino acid composition, amino acid sequence, or protein concentration analysis according to known methods.
  • synthetic H ⁇ 1 proteins ofthe present invention may be prepared by various synthetic methods of peptide synthesis via condensation of one or more amino acid residues, in accordance with conventional peptide synthesis methods.
  • peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430 A peptide synthesizer (Applied Biosystems, Foster City, CA), according to manufacturer's instructions.
  • Other methods of synthesizing peptides or peptidomimetics are well known to those skilled in the art.
  • the invention relates to compositions and methods for using such polypeptides and polynucleotides for treating diseases associated with increased cellular levels of cholesterol, for instance NP-C2 disease, by administering a HEI gene or protein, in a pharmaceutically acceptable and appropriate delivery vehicle, to increase HEI mediated cholesterol egress from lysosomes and the down-regulation of endogenous cellular production of cholesterol.
  • the compositions and methods ofthe present invention may be used for treating a disease associated with increased lysosomal cholesterol transport by administering an HEI antisense polynucleotide sequence in a pharmaceutically acceptable and appropriate delivery vehicle.
  • compositions ofthe present invention may be formulated and used as tablets, capsules, or elixirs for oral administration; suppositories for rectal or vaginal administration; sterile solutions and suspensions for parenteral administration; creams, lotions, or gels for topical administration; aerosols or insufflations for intratracheobronchial administration; and the like. Preparations of such formulations are well known to those skilled in the pharmaceutical arts. The dosage and method of administration can be tailored to achieve optimal efficacy and will depend on factors that those skilled in the medical arts will recognize.
  • injectable pharmaceuticals may be prepared in conventional forms, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection; or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, or the like.
  • the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e.g. liposomes) may be utilized.
  • a therapeutic agent consisting of an HEI polypeptide or a system capable of expressing this polypeptide, will generally be mixed prior to administration with a not-toxic, pharmaceutically acceptable carrier substance.
  • a not-toxic, pharmaceutically acceptable carrier substance usually, this will be an aqueous solution, such as normal saline or phosphate-buffered saline (PBS), Ringer's solution, lactate-Ringer's solution, or any isotonic physiologically acceptable solution for administration by the chosen means.
  • PBS normal saline or phosphate-buffered saline
  • Ringer's solution such as Ringer's solution, lactate-Ringer's solution, or any isotonic physiologically acceptable solution for administration by the chosen means.
  • the solution is sterile and pyrogen-free, and is manufactured and packaged under current Good Manufacturing Processes (GMP's), as approved by the FDA.
  • GMP's Good Manufacturing Processes
  • the clinician of ordinary skill is familiar with the appropriate ranges for pH, tonicity, and additives or preservatives when formulating pharmaceutical compositions for administration by intravascular injection, intrathecal injection, direct injection into aberrant cells, or by other routes.
  • the effective amount ofthe therapeutic composition to be given to a particular patient will depend on a variety of factors, several of which will be different from patient to patient.
  • a competent clinician will be able to determine an effective amount of therapeutic composition to administer to a patient to effect appropriate cholesterol regulation within a subject's cells. Dosage ofthe therapeutic composition will depend on the treatment ofthe particular disease (e.g., such as NP-C2), route of administration, the nature ofthe therapeutic delivery vehicle, etc.
  • a clinician can determine the maximum safe dose for a subject, depending on the route of administration. For instance, an intravenously administered dose may be more than intrathecally administered dose, given the greater body of fluid into which the therapeutic composition is being administered. Similarly, compositions, which are rapidly cleared from the body, may be administered at higher doses, or in repeated doses, in order to maintain a therapeutic concentration. Utilizing ordinary skill, the competent clinician will be able to optimized the dosage of a particular therapeutic composition in the course of routine clinical trials. [0120]
  • the above components can be utilized to create a pharmaceutical composition consisting of an HEI polypeptide and an acceptable carrier.
  • this HEI polypeptide is substantially similar to SEQ. ID.
  • the HEI polypeptide can be administered directly in the form of purified recombinant protein, to a subject suffering from NP-C2 disease, or it can be expressed within a host cell by presenting the cell with an expression vector containing the HEI polynucleotide sequence. Administration ofthe HEI polypeptide can restore normal cholesterol transport and regulation within a cell by restoring cholesterol egress from the lysosome.
  • the invention also relates to pharmaceutical compositions that include expression systems that contain expression cassettes on vectors that comprise a HEI polynucleotide, or polynucleotides for use in recombinant techniques involving both in vitro and in vivo, as well as ex vivo gene therapy procedures.
  • These expression systems can be utilized to express an HEI polypeptide or polypeptide fragment, HEI RNA sequence, or HEI antisense RNA sequence within a host cell.
  • Expression systems can be utilized to express HEI polypeptide sequences in either prokaryotic and eukaryotic cells, for both investigative and therapeutic purposes.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs ofthe present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides ofthe present invention.
  • Introduction of polynucleotides and polypeptides into host cells can then be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergiffits cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as Fibroblasts, CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes pharmaceutical compositions comprising recombinant constructs that include a HEI DNA, cDNA or RNA sequence.
  • a construct comprises a vector, such as a plasmid or viral vector, into which the clone has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the genetic sequence, transcription initiation sequences and enhances sequences. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phagescript, psiX 174, pbluescript SK, pbs s, pNH8A, pNH 16a, pNHI8A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSN2CAT, pOG44, pXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • cD ⁇ A of human HEI may be inserted in the pEF/myc/cyto vector (from Invitrogen) and/or the pCMV-Tag3b vector (from Stratagene), which can then be used with anti-Myc Ab, to transform Stem, HeLa, Fibroblast (or other) cells with HEI D ⁇ A.
  • the HEI protein may be isolated, purified and directly injected in to the appropriate tissue, infused to blood cells, or delivered in a lyophilized carrier as described above.
  • any other plasmid or vector may be used as long as they are replicable and viable in the host's cells.
  • a complete mammalian transcription unit and a selectable marker can be inserted into a prokaryotic plasmid for use in in vivo or ex-vivo procedures.
  • the resulting vector is then amplified in bacteria before being transfected into cultured mammalian cells or delivered directly to the subject with an acceptable biological carrier as described below.
  • Examples of vectors of this type include pTK2, pHyg and pRSVneo.
  • these plasmids, constructs, cassettes and vectors may be used in both in vivo and ex vivo procedures.
  • ex vivo procedures involve the removal of a host cell, such as a fibroblast cell, from the subject, recombinant manipulation ofthe cell (i.e., transformation, transduction or transfection with a suitable HEI expression system vector), and the re-delivery ofthe cell back into a host's environment.
  • recombinant HEI DNA, cDNA or RNA may be directly injected to fibroblasts for the production of HEI endogenously.
  • the polynucleotide sequence coding for the antisense sequence encoding the protein RNA may be directly injected to fibroblasts for the endogenous inhibition of HEI.
  • HEI DNA, cDNA, RNA or polynucleotide sequences coding for the antisense sequence encoding the protein may also be delivered using other appropriate means, including vectors, as described, and well known in the recombinant arts.
  • a wide variety of recombinant plasmids may be engineered to expre'ss the HEI protein and used to deliver HEI to a cell. These include the use of naked DNA and HEI plasmids to directly transfer genetic material into a cell (Wolfe et al., 1990); formulations of HEI encoding trapped liposomes (Ledley et. al., 1987) or in proteoliposomes that contain other viral envelope receptor proteins (Nicolau et al., 1983); and HEI -encoding DNA, or antisense sequence, coupled to a polysineglycoprotein carrier complex.
  • the HEI encoding sequence may be incorporated as part of an expression cassette, a nucleotide sequence that contains at least one coding sequence along with sequence elements that direct the initiation and termination of transcription.
  • An expression cassette may include additional sequences, including but not limited to promoters, enhancers, and sequences involved in post-transcriptional or post-translational processes.
  • methods for the delivery of nucleotide sequences to cells are well known in the recombinant arts.
  • Such methods for in vitro delivery further include, but are not limited to: microinjection, calcium phosphatase, lyposomes, and electroporation.
  • Genetic material such as HEI nucleotides ofthe present invention, may be delivered to cells, in vivo or ex vivo, using various different plasmid based delivery platforms, including but not limited to recombinant ADV (such as that described in U.S. Pat. No. 6,069,134 incorporated by reference herein), AAV (such as those described by U.S. Pat. No. 5,139,941 incorporated by reference herein), MMLV, Herpes Simplex Virus (U.S. Pat. No. 5,288,641, incorporated by reference herein), cytomegalovirus, lentiviral, and overall, retroviral gene delivery systems, well known and practiced with in the art.
  • ADV such as that described in U.S. Pat. No. 6,069,134 incorporated by reference herein
  • AAV such as those described by U.S. Pat. No. 5,139,941 incorporated by reference herein
  • MMLV Herpes Simplex Virus
  • cytomegalovirus lentivi
  • These systems typically include a plasmid vector including a promoter sequence (such as CMV early promoter) operably linked to the nucleotide coding the gene of interest (inserted into an appropriate gene insertion site; i.e., an IRES site), as well as transcription initiation sequences, enhancer sequences, a terminating signal (such as a Poly-A tail i.e., BGH), and the appropriate mutations so as to make the delivery vehicle replication defective (e.g., Psi sequence deletions) and safe for therapeutic uses.
  • a promoter sequence such as CMV early promoter
  • an IRES site i.e., an IRES site
  • transcription initiation sequences such as a Poly-A tail i.e., BGH
  • BGH Poly-A tail i.e., BGH
  • the construction ofthe appropriate elements in a vector system containing the nucleotides ofthe present invention is well within the skills of one versed in the recombinant arts.
  • vector and/or expression systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia, viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • chromosomal, episomal and virus-derived systems e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any delivery system such as a vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used.
  • the appropriate HEI nucleotide sequence may be inserted into delivery system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, Molecular Cloning, A Laboratory Manual (supra).
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol acetyl transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
  • bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1. Selection ofthe appropriate vector and promoter is well within the level of ordinary skill in the art. [0132] Administration ofthe HEI polypeptide, or a vector capable of expressing the HEI polypeptide, can be utilized to treat diseases involving faulty cholesterol transport and regulation. A vector containing an HEI genetic element can be used in conjunction with a biologically acceptable carrier to express HEI in a host cell.
  • such the HEI genetic element in such an expression system comprises a polynucleotide sequence with at least 70% similar to SEQ. ID. NO: 1.
  • another HEI genetic element such as HEI RNA, cDNA, or antisense polynucleotide can be used instead.
  • the HEI genetic element is contained within an expression cassette.
  • Administration ofthe HEI expression system to a cell can serve to restore both lysosomal cholesterol transport and endogenous cholesterol regulation. Expression of HEI within a host cell can thus potentially ameliorate not only NP-C2 disease, but any disease in which cholesterol regulation is faulty.
  • the HEI polypeptide is to be expressed for use in screening assays, generally, the protein is produced intracellularly requiring the cells first to be lysed before the polypeptide is recovered.
  • the HEI polypeptides can be recovered and substantially purified from recombinant cell cultures by well known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification.
  • HEI screening assays can be utilized to screen for both NP-C2 and NP-C1 diseases. Patients with NP-C2 disease have either a homozygous or heterozygous mutation in both copies ofthe gene encoding for HEI. Detection of a mutation in the HEI gene sequence of SEQ. ID. NO: 1 can be utilized to diagnose NP-C2 disease. This test can be performed prenatally by analyzing the HEI sequence of a fetus. HEI sequence analysis can also be utilized to diagnose the ability of a subject to genetically transmit NP- C2 disease.
  • Mutations in the HEI gene can consist of deletions, substitutions, splice mutations, or insertions. Mutations can be detected by PCR amplification of a subject's DNA, followed by sequence analysis ofthe HEI gene. Alternatively, mutations in the HEI gene can be detected by hybridizing the HEI gene sequence of a subject with a labeled polynucleotide probe complementary to the HEI gene sequence of SEQ. ID. NO: 1.
  • this polynucleotide probe will consist of a DNA molecule bound to a label selected from radioactive isotopes (e.g. P, I, S), biotin, an enzyme reporter group (e.g. horseradish peroxidase, alkaline phosphatase), a chemiluminescent label, a fluorescent label, or an antibody.
  • a label selected from radioactive isotopes (e.g. P, I, S), biotin, an enzyme reporter group (e.g. horseradish peroxidase, alkaline phosphatase), a chemiluminescent label, a fluorescent label, or an antibody.
  • the target HEI gene sequence may be amplified by PCR.
  • Target sequence and probe may be hybridized on a solid support such as a filter membrane, in solution, in situ, or via Southern blot.
  • a battery of probes may be utilized to ensure hybridization between target and probe for any potential mutation.
  • HEI screening can serve as a method of diagnosing NP-C1 disease.
  • Patients with NP-C 1 exhibit raised levels of HEI expression.
  • a comparison of HEI protein or mRNA levels in a cell as compared to a healthy control cell can be used to diagnose the presence of NP-C1 disease.
  • Increased HEI expression can be detected by measuring HEI mRNA levels using techniques such as Northern blot, RT-PCR, or ribonuclease protection assay, or by measuring HEI protein levels using techniques such as Western blotting.
  • Retrovimses have promise as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992, incorporated herein by reference).
  • a third method uses other viruses, such as adenovirus, herpes simplex virues (HSV), cytomegalovirus (CMV), and adeno-associated virus (AAV), which are engineered to serve as vectors for gene transfer.
  • viruses such as adenovirus, herpes simplex virues (HSV), cytomegalovirus (CMV), and adeno-associated virus (AAV)
  • HSV herpes simplex virues
  • CMV cytomegalovirus
  • AAV adeno-associated virus
  • adenovirus gene transfer systems may be used. Such a system is based upon recombinant, engineered adenovirus which is rendered replication-incompetent by deletion of a portion of its genome, such as El, and yet still retains its competency for infection.
  • adenoviruses deleted in both El and E3 regions are capable of carrying up to 10 Kb of foreign DNA and can be grown to high titers in 293 cells (Stratford-Perricaudet and Perricaudet, 1991a). Surprisingly persistent expression of transgenes following adenoviral infection has also been reported.
  • the recombinant HEI DNA, cDNA or RNA may be delivered to cells, for the production of HEI endogenously, and the polynucleotide sequence coding for the antisense sequence encoding the protein RNA, may be delivered to cells, for the endogenous inhibition of HEI, by use of biologically compatible carriers or excipients. This may be useful in inducing or inhibiting intracellular cholesterol levels and cholesterol transport.
  • Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences (A. P. Gennaro, ed.; Mack, 1985). For example, sterile saline or phosphate- buffered saline at physiological pH may be used.
  • Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid may be added as preservatives.
  • Antioxidants and suspending agents may also be used.
  • the dosage and method of administration can be tailored to achieve optimal efficacy and will depend on factors that those skilled in the medical arts will recognize.
  • injectable pharmaceuticals may be prepared in conventional forms, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection; or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, or the like.
  • the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like.
  • absorption enhancing preparations e.g. liposomes may be utilized.
  • a preferred embodiment ofthe present invention involves methods for the treatment of diseases associated with an unhealthy increase or decrease of intracellular levels of cholesterol in a subject. These methods involve administering to the subject a pharmaceutical composition that includes an effective amount of a HEI protein or a nucleotide sequence coding for the HEI protein or a nucleotide sequence that codes for the anti-sense sequence ofthe nucleotide sequence coding for the HEI protein. These may be delivered by suitable means, as described above, including the use of vectors and or acceptable biological carriers. [0142] The foregoing is intended to be illustrative ofthe embodiments ofthe present invention, and are not intended to limit the invention in any way.
  • Example 1 Analysis of human brain mannose 6-phosphorylated glycoproteins [0143] Identification of HEI as the gene responsible for NP-C2 disease arose from an ongoing study directed at characterizing the lysosome proteome. The approach of this study was based on the fact that many soluble lysosomal proteins acquire a posttranslational modification that distinguishes them from most other types of proteins, the mannose 6-phosphate (Man6-P) marker. This modification is recognized by Man6-P receptors (MPRs), which divert newly synthesized lysosomal enzymes from the secretory pathway to the endolysosomal system (33). Purified MPR derivatives typically bind phosphorylated lysosomal proteins with subnanomolar affinity and can be used to detect and purify Man6-P glycoproteins (32, 34).
  • MPRs Man6-P receptors
  • MPR affinity-purified proteins from the human brain were fractionated by two- dimensional electrophoresis with precast gels (Invitrogen, Carlsbad, CA). The first dimension consisted of isoelectric focusing on pH 3 to 10 gels without prior sample reduction, while the second dimension consisted of sample reduction by SDS-PAGE on 10 to 20%) polyacrylamide gels.
  • the resultant two-dimensional gel map of MPR affinity- purified proteins contained a group of proteins sharing the same NH 2 -terminal sequence, as determined by Edman degradation, with these sequences likely representing differentially glycosylated isoforms ofthe same protein.
  • FIG. IB A nitrocellulose membrane containing 5 ⁇ g ofthe MPR affinity-purified proteins, pictured in Figure IB, was probed with these antibodies. These Western blot results confirmed that the MPR affinity-purified proteins containing the HEI NH 2 -terminus sequence were indeed HEI.
  • Figure IC again illustrates a nitrocellulose membrane containing 5 ⁇ g ofthe MPR affinity-purified proteins, this time probed with radiolabeled MPR. Probing with this 2 nM 125 I-labeled soluble cation-independent MPR confirmed that HEI contained the Man6- P modification. Inclusion of 10 mM Man6-P during probing abrogated the signal.
  • Northern blot analysis was performed to measure the level of HEI expression in various human tissues. 2 ⁇ g of polyadenylated RNA from various human tissues (Origene Technologies, Rockville, MD) was immobilized on a membrane and probed with a 32 P- labeled DNA fragment corresponding to the entire coding region of HEI . This Northern blot, pictured in Figure ID, revealed a single transcript of 0.9 kb in all tissues examined. The highest levels of HEI expression were observed in testis, kidney, and liver, while the lowest levels of expression were observed in lung and muscle.
  • Example II Subcellular distribution of HEI homolog in rat liver [0148] Rat liver homogenate was subjected to differential centrifugation as described in (39). Differential centrifugation resulted in the homogenate differentiation into five fractions: N, the nuclear fraction; M, the heavy mitochondrial fraction; L, the light mitochondrial fraction; P, the microsomal fraction; and S, the soluble fraction. Each fraction was assayed for the presence of both HEI and lysosomal protein activity.
  • Figure 2A illustrates the results of these assays. The upper panel represents Western blot analysis of each fraction with rabbit antibodies to HEI. After differential centrifugation, HEI was found mainly in the heavy mitochondrial (M) fraction and the light mitochondrial (L) fraction.
  • the lower panel of Figure 2A illustrates lysosomal protein activity in each fraction.
  • the filled bars represent ⁇ -galactosidase activity
  • the open bars represent tripeptidyl peptidase activity
  • the hatched bars represent ⁇ -hexosaminidase activity.
  • the highest levels of lysosomal protein activity were observed in the heavy mitochondrial (M) fraction and the light mitochondrial (L) fraction.
  • the presence of HEI only in the fractions with the highest lysosomal protein activity showed that HEI itself is a lysosomal protein.
  • the upper panel represents Western blot analysis of each sucrose gradient fraction using rabbit antibodies to HEI, while the lower panel represents lysosomal protein activity. Codistribution of HEI and lysosomal protein activity was once again observed, with both shifting to earlier fractions in the sucrose gradient. The results of these isopycnic centrifugation experiments verify that most, if not all, HEI is located in lysosomes.
  • Example III Measurement of HEI levels in control and mutant fibroblasts
  • Fibroblasts originated from either the Coriell collection (designated by GM numbering) or from Peter Pentchev (designated by NPC numbering).
  • Control fibroblasts were from cells lines GM06556, GM05757B, and GM03625F.
  • Mutant fibroblasts originated from subjects having either sea blue histiocyte disease, NP-C1 disease, or NP- C2 disease. Sea blue histiocyte fibroblasts were from cell lines GM01912 and GM00843.
  • NP-C1 disease fibroblasts were from cell lines GM11095, GM03123A, GM00110B, and NPC1 90.48.
  • NP-C2 disease fibroblasts were from cell lines NPC2 93.10 and NPC2 99.04.
  • Fibroblasts were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum. The cells were lysed in 0.1% Triton X-100 and 150 mM NaCl, and extracts were centrifuged 20 minutes at 13,000g. Soluble protein (7 ⁇ g) was separated by SDS- polyacrylamide gel electrophoresis. The blot was probed with rabbit antibodies to HEI and chemilummescence.
  • the blot was reprobed with rabbit antibodies to cathepsin D (Calbiochem, LaJolla, CA) and chemilummescence.
  • cathepsin D Calbiochem, LaJolla, CA
  • the results of these blots are pictured in Figure 3A.
  • the top panel represents Western blot analysis of HEI, while the lower panel represents Western blot analysis ofthe cathepsin D control.
  • Lanes 1, 2, and 3 represent control fibroblast samples
  • lanes 4 and 5 represent sea blue histiocyte disease samples
  • lanes 6, 7, 8, and 9 represent NP-Cl disease samples
  • lanes 10 and 11 represent NP-C2 disease samples.
  • HEI expression was undetectable in fibroblasts from the NP-C2 patients.
  • HEI expression was detectable in control fibroblasts as well as fibroblasts taken from patients with sea blue histiocyte disease and NP-Cl disease.
  • Example IN Sequence analysis of HEI gene in ⁇ P-C2 patients
  • the complete sequence ofthe human HEI gene is available under GenBank accession number AC005479.
  • a schematic ofthe HEI gene and protein is presented in Figure 3B.
  • ntl represents the first nucleotide ofthe initiation codon.
  • the arrowheads represent potential N-linked glycosylation sites, with the filled arrowhead denoting a site that is conserved among mammalian HEI orthologs.
  • the predicted disulfide pairing ofthe cysteines portrayed in the schematic is assigned by homology to equivalent cysteines in an apparently related dust mite protein (42).
  • the genomic structure of HEI was determined by sequence alignments between the HEI cDNA (Q15668) and genomic DNA sequence (AC005479).
  • the chromosomal localization of HEI 14q24.3 was determined by identifying mapped clones in the Sequence Tagged Sites (STS) database that aligned to AC005479 (e.g. STS clones G38283, G38146, and G38077) and was confirmed by radiation hybrid panel mapping to chromosome 14 with the Coriell monochromosomal somatic hybrid panel.
  • STS Sequence Tagged Sites
  • the human HEI gene sequence was used to design polymerase chain reaction primers for amplifying the entire HEI coding region. Sequence analysis ofthe amplified HEI gene sequence revealed the presence of mutations in two unrelated NP-C2 patients. The location of these two mutations is designated by the dark arrows above the protein schematic in Figure 3B.
  • One patient (NPC2 99.04) was homozygous for a transversion of G to T in exon 1 that results in conversion of amino acid E20 to a termination codon. Since E20 corresponds to the NH -terminus ofthe mature protein, this represents a null mutation.
  • the other patient (NPC2 93.10) was compound heterozygous for the Glu20Stop mutation and a single nucleotide deletion in exon 2 that shifts the reading frame and generates a stop codon four codons downstream. This severe truncation also affects the predicted disulfide pairing ofthe protein and is likely to represent a null allele. No mutations were detected in sequence analysis of DNA from eight individuals who represented either unaffected controls, patients with NP-Cl, or patients with other diseases. These findings illustrate that mutations in HEI are specifically associated with NP-C2.
  • a fragment corresponding to human HEI cDNA flanked by JKhol sites was generated using standard PCR-based methods and subcloned into - ⁇ b ⁇ l-digested expression vector pMSXNDl, yielding a construct that contains an HEI expression cassette, a neomycin-resistance cassette for G418 selection, and a dihydrofolate reductase expression cassette for MTX-based selection. After restriction mapping, correctly oriented constructs were sequenced to confirm absence of unwanted changes in the coding region. Plasmid DNA was linearized with Pvul before transfection.
  • CHO cells were maintained in Dulbecco's modified Eagle's (DME)/F12 medium (Sigma) supplemented with 10%) (vol/vol) fetal-bovine serum (FBS) and were stably transfected using LIPOFECTAMINETM (Gibco).
  • DME Dulbecco's modified Eagle's
  • FBS fetal-bovine serum
  • LIPOFECTAMINETM LIPOFECTAMINETM
  • Figure 4D represents a Western blot comparing HEI expression in the HEI -transfected CHO cells (lane 2) to HEI expression in normal CHO cells (lane 1).
  • HEI secreted from transfected CHO cells is secreted as a functional protein. Presumably, the overexpressed HEI overwhelms the sorting machinery ofthe cell, resulting in secretion rather than lysosomal targeting.
  • the secreted HEI contains the mannose 6-phosphate modification, meaning it can be delivered by receptor mediated endocytosis and subsequently targeted to the lysosome.
  • Control NP-C2 fibroblasts were cultured for 4 days in RPMI 1640 medium (Gibco) supplemented with 15% FBS and maintained at 37 °C in a humidified atmosphere containing 5%> CO 2 .
  • Experimental cells were cultured in the same manner, but were treated with a supplement consisting of either 0.3%o conditioned medium from the HEI transfected CHO cells or 0.3% conditioned medium from untransfected CHO cells. Accumulation of cholesterol in the NP-C2 fibroblasts was then demonstrated by punctate fluorescence after probing with filipin, a cholesterol antibiotic.
  • Figure 4A illustrates filipin staining results for control NP-C2 fibroblasts
  • Figure 4B and 4C illustrate filipin staining results for NP-C2 fibroblasts treated 0.3%) medium from HEI producing CHO cells or normal CHO cells, respectively.
  • cultivation of NP-C2 cells with a small amount (0.3%> vol/vol) of medium from the CHO cells transfected with HEI diminished cholesterol accumulation compared with controls (naive medium or equivalent amounts of conditioned medium from untransfected CHO cells).
  • NP-C2 cells Cultivation of NP-C2 cells with large amounts (>10%) of conditioned media from untransfected CHO cells partially reversed cholesterol accumulation, presumably reflecting the presence of low amounts of endogenous HEI homolog secreted by the CHO cells.
  • comparable experiments showed that the HE1- conditioned medium had no effect on reducing cholesterol accumulation in NP-Cl fibroblasts, thus further demonstrating the specificity of the defect inNP-C2.
  • the NP-C2 fibroblasts were grown to confluence, and fields containing approximately 200 cells were selected under bright-field illumination to eliminate operator bias. Fluorescence measurements were then collected with charge- coupled device camera. For each condition, the average pixel intensity of five fields was used for the analysis. Data were corrected for background staining obtained with unaffected control fibroblasts (477 ⁇ 15, mean ⁇ standard error). These results are pictured in Figure 4E, with A representing control NP-C2 fibroblasts, B representing NP- C2 fibroblasts treated with medium from HEI producing CHO cells, and C representing NP-C2 fibroblasts treated with medium from normal CHO cells. The asterisk indicates that the difference in staining intensity ofthe HEI -treated fibroblasts compared with the two control groups is statistically significant (R ⁇ 0.05).
  • NP-C2 disease is due to a deficiency in a soluble lysosomal protein is consistent with earlier observations that cocultivation of mononuclear NP-Cl and NP-C2 fibroblasts partially reversed cholesterol accumulation in a subset ofthe cells (11). It may also explain why fibroblasts from patients with I-cell disease, which lack the enzyme that normally generates the Man6-P lysosomal targeting signal and have low intracellular levels of multiple lysosomal proteins, accumulate LDL-derived cholesterol (36).
  • Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis. J. Biol. Chem. 271 :19191-19198.
  • ⁇ iemann- Pick Cl is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network. Mol. Genet. Metab. 68:1-13.

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Abstract

La maladie de Niemann-Pick de type C2 (NP-C2) est une anomalie létale du stockage des lipide caractérisée par une accumulation lysomiale massive du cholestérol et la présente invention identifie le HE1 comme gène responsable de la NP-C2. Le traitement des fibroblastes de la NP-C2 par un élément génétique d'HE1 exogène a permis d'améliorer le phénotype d'accumulation du cholestérol, le HE1 influant sur le transport intracellulaire du cholestérol. L'invention porte sur des compositions thérapeutiques consistant en séquences de polynucléotides et de polypeptides du HE1, ainsi que sur un système d'expression du HE1 dans des cellules cibles. Lesdites compositions thérapeutiques peuvent servir à cibler des maladies dues à un mauvais transport et une mauvaise régulation du cholestérol, dont la NP-C2, l'athérosclérose, la maladie d'Alzheimer, le diabète, et les maladies cardio-vasculaires. L'invention porte également sur des procédés de diagnostic à la fois de la NP-C2, et de la capacité d'un sujet à transmettre la maladie, par détection des mutations de la séquence du gène du HE1.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1394267A1 (fr) * 2002-08-19 2004-03-03 Bayer HealthCare AG Polymorphismes à nucléotide unique permettant de pronostiquer les maladies cardio-vasculaires, les effets secondaires et l'efficacité des medicaments
WO2023141676A1 (fr) * 2022-01-28 2023-08-03 The University Of Melbourne Arnm thérapeutiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0440321A2 (fr) * 1990-02-01 1991-08-07 IHF INSTITUT FÜR HORMON- UND FORTPFLANZUNGSFORSCHUNG GmbH Séquences d'ADN spécifique d'épididymis et leur utilisation
WO1999001555A1 (fr) * 1997-07-03 1999-01-14 The United States Of America Represented By The Secretary, Department Of Health And Human Services Genes de la maladie de niemann-pick de type c

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0440321A2 (fr) * 1990-02-01 1991-08-07 IHF INSTITUT FÜR HORMON- UND FORTPFLANZUNGSFORSCHUNG GmbH Séquences d'ADN spécifique d'épididymis et leur utilisation
WO1999001555A1 (fr) * 1997-07-03 1999-01-14 The United States Of America Represented By The Secretary, Department Of Health And Human Services Genes de la maladie de niemann-pick de type c

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
C. KIRCHHOFF ET AL: "Molecular cloning of HE1, a major secretory protein of the human Epididymis" BIOLOGY OF REPRODUCTION, vol. 54, 1996, pages 847-856, XP008015206 *
MILLAT GILLES ET AL: "Niemann-Pick C1 disease: The I1061T substitution is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype." AMERICAN JOURNAL OF HUMAN GENETICS, vol. 65, no. 5, November 1999 (1999-11), pages 1321-1329, XP002235643 ISSN: 0002-9297 *
MILLAT GILLES ET AL: "Niemann-Pick disease type C: Spectrum of HE1 mutations and genotype/phenotype correlations in the NPC2 group." AMERICAN JOURNAL OF HUMAN GENETICS, vol. 69, no. 5, November 2001 (2001-11), pages 1013-1021, XP002235646 ISSN: 0002-9297 cited in the application *
NAURECKIENE SAULE ET AL: "Identification of HE1 as the second gene of Niemann-Pick C disease." SCIENCE (WASHINGTON D C), vol. 290, no. 5500, 22 December 2000 (2000-12-22), pages 2298-2301, XP002235645 ISSN: 0036-8075 cited in the application *
OKAMURA NAOMICHI ET AL: "A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol." BIOCHEMICA ET BIOPHYSICA ACTA, vol. 1438, no. 3, 10 June 1999 (1999-06-10), pages 377-387, XP002235644 ISSN: 0008-3002 *
STEINBERG STEVEN J ET AL: "Complementation studies in Niemann-Pick disease type C indicate the existence of a second group." JOURNAL OF MEDICAL GENETICS, vol. 31, no. 4, 1994, pages 317-320, XP008015357 ISSN: 0022-2593 *
VANIER M T ET AL: "Genetic heterogeneity in Niemann-Pick disease: A study using somatic cell hybridzation and linkage analysis" AMERICAN JOURNAL OF HUMAN GENETICS, UNIVERSITY OF CHICAGO PRESS, CHICAGO,, US, vol. 58, no. 1, 1996, pages 118-125, XP002078615 ISSN: 0002-9297 *
YAMAMOTO TOSHIYUKI ET AL: "Genotype-phenotype relationship of Niemann-Pick disease type C: A possible correlation between clinical onset and levels of NPC1 protein in isolated skin fibroblasts." JOURNAL OF MEDICAL GENETICS, vol. 37, no. 9, September 2000 (2000-09), pages 707-711, XP008015193 ISSN: 0022-2593 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1394267A1 (fr) * 2002-08-19 2004-03-03 Bayer HealthCare AG Polymorphismes à nucléotide unique permettant de pronostiquer les maladies cardio-vasculaires, les effets secondaires et l'efficacité des medicaments
WO2004018709A2 (fr) * 2002-08-19 2004-03-04 Bayer Healthcare Ag Polymorphismes simple nucleotide utilises pour la prediction sensible d'effets indesirables et de l'efficacite d'un medicament
WO2004018709A3 (fr) * 2002-08-19 2004-10-28 Bayer Healthcare Ag Polymorphismes simple nucleotide utilises pour la prediction sensible d'effets indesirables et de l'efficacite d'un medicament
WO2023141676A1 (fr) * 2022-01-28 2023-08-03 The University Of Melbourne Arnm thérapeutiques

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