WO2002059369A2 - Compositions and methods for the treatment of diseases related to faulty cholesterol regulation - Google Patents

Abstract

Niemann-Pick type C2 (NP-C2) disease is a fatal lipid storage disorder characterized by massive lysosomal accumulation of cholesterol. The present invention identifies HE1 as the gene responsible for NP-C2. Treatment of NP-C2 fibroblasts with an exogenous HE1 genetic element ameliorated the cholesterol accumulation phenotype. HE1 functions in intracellular cholesterol transport. The present invention provides therapeutic compositions consisting of HE1 polynucleotide and polypeptide sequences, as well as an expression system for expressing HE1 in target cells. These therapeutic compositions can be used to target diseases involving faulty cholesterol transport and regulation, including NP-C2, atherosclerosis, Alzheimer's, diabetes, and cardiovascular disease. In addition, the present invention provides methods of diagnosing both NP-C2 and the ability of a subject to genetically transmit the disease by detecting mutations in the HE1 gene sequence.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF DISEASES RELATED TO FAULTY CHOLESTEROL REGULATION

BACKGROUND OF THE INVENTION [0001] The present utility patent application claims priority to provisional patent application U.S. Ser. No. 60/245,031 (Lobel), filed November 1, 2000, the disclosure of which is incorporated by reference in its entirety herein.

GOVERNMENT INTEREST [0002] This invention was made with government support by the following Public Health Service grants: DK54317, NS37918, and DK45992 from the National Institute of Health. The government may own certain rights in the present invention.

FIELD OF THE INVENTION [0003] The present invention relates to the field of molecular biology, cholesterol storage disease, cholesterol regulation, and cardiovascular disease. In particular, 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.

BACKGROUND [0004] Niemann-Pick disease is a general term for a group of diseases that affect metabolism. The three most common recognized forms ofthe disease are types A, B, and C (NP-A, NP-B, and NP-C). NP-A and NP-B are autosomal recessive disorders linked to a deficiency in the enzyme sphingomyelinase (ASM) (1). In a subject with NP-A or NP- B, ASM does not properly metabolize sphingomyelin, leading to an accumulation of this lipid within the cell (2). Although 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).

[0005] Niemann-Pick disease type C (NP-C) is an autosomal recessive lipid storage disorder characterized by progressive deterioration ofthe central nervous system, visceral symptoms, and premature death (3). There are two forms of NP-C disease, NP-C1 and NP-C2. NP-C1 is substantially more common than NP-C2, with a rate of occurrence estimated at 1 in 10⁵ live births, as opposed to an occurrence of only 8 lαiown cases of NP- C2 worldwide. At the cellular level, 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). In addition, 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).

[0006] In a healthy subject, 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

cellular sites (8).

[0007] 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.

[0008] 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.

[0009] The gene responsible for NP-C 1 disease has been cloned and characterized (12). NPC1 encodes an integral transmembrane protein consisting of 1278 amino acids, with a lysosomal targeting motif and a putative sterol-sensing domain (12). Despite extensive investigation, the precise function of NPC1 remains elusive. NPC1 resembles a family of bacterial permeases that transport various substances, including fatty acids, through the bacterial cell membrane (13). There is evidence that NPC1 has a permease-like pump function, but the implication for cholesterol transport is unclear (13). [0010] NP-C2 is extremely rare, with only eight total cases having been reported worldwide (14). The cases reported thus far arose in France, Algeria, Italy, Germany, the Czech republic, and Turkey (14). One of these cases exhibited a milder phenotype, with the patient displaying juvenile onset of neurological symptoms and prolonged survival (14). In the other seven cases, patients suffered a severe and rapid disease course, with all patients dying between 6 months and 4 years of age (14). Six of these seven cases displayed pronounced lung involvement, with patients dying of respiratory failure (14). Two of these patients also developed severe neurological disease during infancy (14). There is currently no available therapy for the treatment of NP-C2. The devastating nature ofthe disease, along with its apparent link to the crucial process of intracellular cholesterol transport, make the need for therapeutic development extremely important. [0011] 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). In addition, a bovine homolog is present in milk (22) and bovine and murine messages are detected in several tissues (22, 23). These observations suggest that, in addition to its speculated role in sperm maturation (19, 24), HEI may have a more global function. 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).

SUMMARY OF THE INVENTION [0012] In summary, Niemann-Pick disease type C2 (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. The present inventor has discovered that HEI, a previously described cholesterol binding protein isolated from epididymal fluid, is 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). Further, the present inventor has discovered that a defect in HEI protein expression is responsible for the accumulation of LDL-derived cholesterol in P- C2 patients. Based on the work ofthe present inventor, it was determined that HEI is undetectable in fibroblasts from NP-C2 patients, but present at normal levels in control patients. In addition, HEI levels are elevated in NP-Cl patients. These observations led the present inventor to develop a treatment for subjects affected by NP-C2, and as described herein below, the treatment of NP-C2 (HEI deficient) fibroblasts with exogenous recombinant HEI protein ameliorates accumulation of LDL-derived cholesterol.

[0013] The exact role of HEI in cholesterol transport has still not been fully characterized. The present inventor suggests that one role for HEI can be as a carrier that prevents unregulated intercalation of cholesterol into lipid bilayers, selectively transferring sterols to transmembrane proteins such as NPC1. By facilitating this transfer, HEI ensures that free LDL-derived cholesterol can escape from the lysosomal compartment. This mechanism explains why NP-C2 cells, in which HEI is inactive, exhibit large-scale cholesterol accumulation.

[0014] Hence, although the role of normal HEI in cholesterol transport is still not completely clear, the present discovery identifies defective HEI expression as the cause of NP-C2, and thus implicates this defective expression in faulty cholesterol transport. This discovery ofthe molecular basis for NP-C2 facilitates genetic, biochemical, and physical studies to further elucidate the process and mechanism of lysosomal cholesterol transport. [0015] In addition, discovery ofthe role of 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. In Alzheimer's disease, high cholesterol levels appear to increase formation of β-amyloid protein, which has been implicated as the primary cause of brain degeneration (29). Research with both cultured cells and animals has shown that statins (drugs designed to lower cholesterol levels) appear to lower the prevalence of Alzheimer's disease. However, statins are not an ideal therapy for decreasing Alzheimer's, due to their sometimes dangerous side effects, which include liver damage and rhabdomyolosis (29). In 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. [0016] The discovery of 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. In addition, 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.

[0017] According to one aspect of this invention, a pharmaceutical composition consisting ofthe polynucleotide encoding HEI is provided. Preferably, 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. Preferably, this polypeptide sequence is identical to the sequence of SEQ. ID. NO: 2, or is a sequence substantially similar to SEQ. ID. NO: 2. In addition, 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.

[0018] 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. Preferably, the delivery vehicle can be a plasmid, cosmid, bacteriophage, or virus, and includes all the appropriate, additional regulatory elements. [0019] The present invention may be implicated not only in NP-C2, but also in other diseases involving faulty cholesterol regulation. Hence, 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. Preferably, 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. In one preferred embodiment, this delivery vehicle may be a vector such as a plasmid, cosmid, or virus, which may contain one or more additional elements. [0020] Further, 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. Preferably, 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. NO: 1. In addition to providing a means of diagnosing NP-C2 in a patient, 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.

BRIEF DESCRIPTION OF THE DRAWINGS [0021] The invention is best understood from the following detailed description when read in connection with the accompanying drawings, in which: [0022] FIG. 1. Analysis of human brain mannose 6-phosphorylated glycoproteins. A.

Coomassie blue-stained polyvinylidine difluoride membrane. B. Nitrocellulose membrane probed with antibodies to HEI and visualized by chemiluminescence. C.

Nitrocellulose membrane probed with 2nM ¹²⁵I-labeled soluble cation-independent MPR.

D. HEI expression in various human tissues.

[0023] 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.

[0024] FIG. 3. A. HEI and cathepsin D protein levels in control and mutant fibroblasts.

I

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.

[0025] 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.

DETAILED DESCRIPTION Introduction

[0026] Applicants have identified HEI as the gene responsible for Niemann-Pick type C2 disease. Niemann-Pick disease type C (NP-C) is an autosomal recessive lipid storage disorder characterized by progressive deterioration of the central nervous system, visceral symptoms, and premature death (3). There are two forms of NP-C disease, NP-C1 and NP-C2. NP-C1 is substantially more common than NP-C2, with a rate of occurrence estimated at 1 in 10⁵ live births, as opposed to an occurrence of only 8 known cases of NP- C2 worldwide. At the cellular level, the most prominent feature ofthe NP-C lesion is lysosomal sequestration of low density lipoprotein (LDL)-derived cholesterol, resulting in downstream effects on cholesterol homeostasis (4, 30). Somatic cell hybridization experiments with skin fibroblast cultures from unrelated NP-C patients demonstrated the existence of a major complementation group comprising ~95% of cases, designated NP- Cl, and a minor complementation group, designated NP-C2 (9, 10, 11). [0027] Discovery ofthe molecular defect responsible for producing NP-C2 arose from a study directed at characterizing the lysosome proteome. The approach that led to the discovery 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 (31, 34). [0028] A two-dimensional gel map of MPR affinity-purified proteins from human brain contained a group of proteins sharing the sameNH₂-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.

[0029] Analysis of HEI mRNA by Northern blotting revealed a single transcript of 0.9 kb in all tissues examined, with highest levels in testis, kidney, and liver and lowest levels in lung and muscle. This wide distribution is consistent with the presence of HEI sequences in numerous cDNA and SAGE libraries [see UniGene cluster Hs.l 19529]. In addition, a bovine HEI homolog is present in milk (22), and bovine and murine HEI mRNAs have been detected in several tissues (22, 23). These observations suggest that, in addition to its postulated specialized role in sperm maturation (19, 24), HEI may have a more global function. Definitions

[0030] Various terms relating to the biological molecules ofthe present invention are used throughout the specification and claims. [0031] "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 including similar activities or improved activities or these activities with decreased undesirable side effects.

[0032] "HEI gene" refers to a polynucleotide as defined above in accordance with the present invention, which encodes an HEI polypeptide.

[0033] 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.

[0034] "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. For example, 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.

[0035] "Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or

DNA. "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. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.

[0036] The term 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. A variety of modifications have been made to DNA and RNA; thus, "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.

[0037] "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.

[0038] 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. See, for instance, Proteins - Structure And Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., "Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in "Postti'anslational Covalent Modification Of Proteins", B, C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al, "Protein Synthesis: Posttranslational Modifications and Aging", Ann AIYAcad Sci (1992) 663:48-62. [0039] "Variant" as the term is used herein, 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.

[0040] The term "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.

[0041] The term "percent identity" or "percent identical" with regards to nucleic acid molecules refers to the percentage ofthe nucleotides ofthe subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program. [0042] "Identity" and "similarity" can be readily calculated by known methods. 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. In preferred methodologies, 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. However, equivalent alignments and similarity/identity assessments can be obtained through the use of any standard alignment software. For instance, the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, WI, and the default parameters used (gap creation penalty=12, gap extension penalty=4) by that program may also be used to compare sequence identity and similarity.

[0043] With respect to single-stranded nucleic acid molecules, 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"). In particular, 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.

[0044] With respect to oligonucleotide constructs, but not limited thereto, 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"). In particular, 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. [0045] The term "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).

[0046] The term "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.

[0047] "Expression system" refers to a system for expressing a recombinant protein within a host cell. Generally, 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). As with the expression cassette, 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. [0048] 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.

[0049] The term "operably linked" or "operably inserted" 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. [0050] 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.

[0051] The terms "promoter", "promoter region", or "promoter sequence" refer 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. Typically, 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. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of

RNA polymerase.

[0052] 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.

[0053] The term "selectable element" refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.

[0054] The term "reporter gene" refers to a gene that encodes a product that is detectable by standard methods, either directly or indirectly.

[0055] 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.

[0056] The term "nucleic acid construct," "DNA construct" or "DNA expression 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". Such a 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.

[0057] 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. Thus, when 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. In another example, 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.

[0058] The term "DNA construct", as defined above, 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. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, 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. This stability is demonstrated by the ability ofthe eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. [0059] 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. [0060] As used herein, the term "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. Preferably the viral mediated delivery system is targeted specifically to the cell, wherein delivery is sought. The production of 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. In light ofthe present disclosure, the skilled artisan will be able to deliver the therapeutic agents ofthe present invention to cells in many different but effective ways. For instance, 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. Naturally, different viral host ranges will dictate the virus chosen for transfer.

[0061] "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. [0062] As used herein, the term "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.

[0063] "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.

[0064] "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.

[0065] The term "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. This includes both the direct delivery of an HEI protein to the cell and the delivery of a gene or DNA segment that encodes HEI, or its antisense polynucleotide sequence, which gene or antisense sequence will direct or inhibit, respectfully, the expression and production of HEI within the cell. Since protein delivery is subject to drawbacks, such as degradation and low cellular uptake, it is contemplated that the use of a recombinant vector that expresses a HEI protein, or encodes for an HEI polynucleotide or antisense sequence, will be of particular advantage for delivery.

[0066] The term "mammal" 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.

[0067] The phrase "therapeutically 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. Polynucleotides

[0068] 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. 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. This mechanism explains why NP-C2 cells, in which HEI is inactive, exhibit large-scale cholesterol accumulation and has led to the development ofthe compositions and methods, herein described below, for the treatment of NP-C2 disease. The summary ofthe invention described above is non-limiting and other features and advantages ofthe invention will be apparent from the following detailed description. [0069] 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. [0070] 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.

[0071] One preferred embodiment ofthe present invention involves 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.

[0072] Hence, 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. In this regard, 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. Also included under 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. [0073] Also included in the present invention are polynucleotides encoding 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.

[0100] 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). 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.

[0102] For instance, 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. In positions of degeneracy where more than one nucleic acid residue could be used to encode the appropriate amino acid residue, 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. [0103] In accordance with the present invention, compositions of nucleic acids having the appropriate level of sequence homology (i.e., 70% identity or greater) with part or all the coding regions of SEQ ID NO:2 may be identified by using hybridization and washing conditions of appropriate stringency. For example, 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 As an illustration ofthe above formula, using [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. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.

[0105] 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

solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42°C, and wash in

2X SSC and 0.5% SDS at 55°C for 15 minutes. A high stringency hybridization is defined

as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured

salmon sperm DNA at 42°C, and wash in IX SSC and 0.5% SDS at 6-5°C for 15 minutes. Very high stringency hybridization is defined as hybridization in 6X SSC, SX Denhardt's

solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42°C, and wash in 0.

1 X SSC and 0.5% SDS at 65°C for 15 minutes.

[0106] The nucleic acids to be used in the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, 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.

[0107] The above components can be utilized to generate a pharmaceutical composition consisting of an effective amount of a purified HΕ1 polynucleotide and an acceptable carrier. Preferably, this polynucleotide has at least 70% similarity with the sequence of SΕQ. ID. NO: 1, and encodes a polypeptide sequence substantially similar to SΕQ. ID. NO: 2. In addition, HΕ1 polynucleotides can comprise the genetic element that is incorporated into an expression vector. Such incorporation allows for the therapeutic expression of HΕ1 in a host cell, which can ameliorate certain disease states in which cholesterol transport and regulation is faulty. HΕ1 polynucleotides that can be utilized in this therapeutic expression system include HΕ1 DNA, cDNA, RNA, or antisense polynucleotides. Antisense HΕ1 polynucleotides can be utilized to downregulate HΕ1, which could be of therapeutic benefit in any disease in which there is excess lysosomal cholesterol transport. Polypeptides

[0108] In one aspect, the present invention relates to pharmaceutical compositions that include human HΕ1 polypeptides (or HΕ1 proteins). The human HΕ1 polypeptides include the polypeptide of SΕQ ID NO:l; as well as polypeptides comprising the amino acid sequence of SΕQ ID NO:l; and polypeptides comprising the amino acid sequences which are substantially similar to the amino acid sequence of SΕQ ID NO:l, that is they have at least 70% identity to that of SΕQ ID NO:l, over its entire length. Preferably HΕ1 polypeptides exhibit at least one biological activity of HΕ1. 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. [0109] 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. Also preferred are 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.

[0110] Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate HEI activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Variants ofthe defined sequence and fragments also form part ofthe present invention. Preferred variants are those that vary from the referents by conservative amino acid substitutions. [0111] The 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. For example, 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.

[0112] According to a preferred embodiment, larger quantities of HEI encoded polypeptide, for use with the present invention, may be produced by expression in a suitable prokaryotic or eukaryotic system. For example, part or all of 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. 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 HΕ1 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 HΕ1 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.

[0114] Using appropriate amino acid sequence information, synthetic HΕ1 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. Preferably, 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, either by solid phase methodologies or in liquid phase, are well known to those skilled in the art. [0115] In particular aspects, 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. Further, 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.

[0116] The pharmaceutical 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.

[0117] When administration is to be parenteral, such as intravenous on a daily basis, 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. In addition, if desired, 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.

[0118] For administration, 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. 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. Preferably, the solution is sterile and pyrogen-free, and is manufactured and packaged under current Good Manufacturing Processes (GMP's), as approved by the FDA. 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. [0119] 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. Utilizing LD₅₀ animal data, and other information available for such administrations, 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. Preferably, this HEI polypeptide is substantially similar to SEQ. ID. NO:l, or to a fragment thereof. 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. Vectors, Host Cells, and Expression

[0121] 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.

[0122] Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs ofthe present invention. For recombinant production, 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.

[0123] Representative examples of appropriate hosts for in vitro procedures include 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.

[0124] More particularly, the present invention also includes pharmaceutical compositions comprising recombinant constructs that include a HEI DNA, cDNA or RNA sequence. Such 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. In a preferred aspect of this embodiment, 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. The following vectors are provided by way of example; 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). As further examples, cDΝA 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 DΝA. Or 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.

[0125] However, any other plasmid or vector may be used as long as they are replicable and viable in the host's cells. In addition, 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. Hence, these plasmids, constructs, cassettes and vectors may be used in both in vivo and ex vivo procedures. As stated, 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. [0126] Further, according to one particular embodiment ofthe present invention, recombinant HEI DNA, cDNA or RNA may be directly injected to fibroblasts for the production of HEI endogenously. While, 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.

[0127] 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. Hence 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.

[0128] 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. [0129] Techniques for preparing replication defective, infective viruses are well known in the art, as exemplified by Ghosh-Choudhury & Graham (9187); McGory et al. (1988); and Gluzman et al. (1982), each incorporated by reference herein. 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. 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.

[0130] A great variety of vector and/or expression systems can be used. Such 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. The expression systems may contain control regions that regulate as well as engender expression. Generally, 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). [0131] 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. Particular named 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. Preferably, such the HEI genetic element in such an expression system comprises a polynucleotide sequence with at least 70% similar to SEQ. ID. NO: 1. However, another HEI genetic element, such as HEI RNA, cDNA, or antisense polynucleotide can be used instead. Preferably, 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.

[0133] If 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. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification. [0134] 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. A subject with a mutation in only one copy ofthe HEI gene will exhibit no signs ofthe disease, but would possess a higher risk of genetically transmitting the disease to their offspring. 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. Generally, 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. To increase the sensitivity ofthe probe, 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. [0135] In addition to NP-C2, HEI screening can serve as a method of diagnosing NP-C1 disease. Patients with NP-C 1 exhibit raised levels of HEI expression. Thus, 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.

[0136] The above-described constructs, cassettes, plasmids, and vectors are useful in gene therapy procedures. Successful gene therapy generally requires the integration of a gene able to correct the genetic disorder into the host genome, where it would co-exist and replicate with the host DNA and be expressed at a level to compensate for the defective gene. Ideally, the disease would be cured by one or a few treatments, with no serious side effects. There are several approaches to gene therapy proposed.

[0137] As described above, basic transfection methods exist in which DNA containing the gene of interest is introduced into cells non-biologically, for example, by permeabilizing the cell membrane physically or chemically. Liposomes or protein conjugates formed with certain lipids and amphophilic peptides can be used for transfection. (Stewart et al., 1992; Torchilin et al., 1992; Zhu et al., 1993, incorporated herein by reference.) This approach is particularly effective in ex vivo procedures involving fibroblasts, which can be temporarily removed from the body and can tolerate this mode of treatment. [0138] A second, transduction approach, capitalizes on the natural ability of viruses to enter cells, bringing their own genetic material with them. For example, 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).

[0139] 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. Although some viruses that can accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect, these viruses have been demonstrated to successfully effect gene expression. For example, 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. Relatively large foreign proteins can be expressed when additional deletions are made in the adenovirus genome. For example, 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.

[0140] Further, 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. For example, sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid may be added as preservatives. Antioxidants and suspending agents may also be used. As stated above, 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. When administration is to be parenteral, such as intravenous on a daily basis, 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. In addition, if desired, 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.

[0141] As described above and exemplified below, 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. Although the invention has been described with respect to specific modifications, the details thereof are not to be construed as limitations, for it will be apparent that various equivalents, changes and modifications may be resorted to without departing from the spirit and scope thereof and it is understood that such equivalent embodiments are to be included herein. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

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).

[0144] 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 NH₂-terminal sequence, as determined by Edman degradation, with these sequences likely representing differentially glycosylated isoforms ofthe same protein. The longest of these sequences consisted ofthe amino acid sequence EPVQFKDXGSVDGVIK, which matches perfectly the processed NH -terminus of HEI. The group of proteins sharing this NH -terminal sequence is circled in the Coomassie blue-stained polyvinylidine difluoride membrane in Figure 1A.

[0145] Based on this amino acid sequence similarity, Western blot experiments were run to confirm that the MPR affinity-purified proteins did indeed represent differentially glycosylated versions of HEI. COOH-terminally hexahistidine-tagged HEI was expressed in Escherichia coli. The expressed protein formed inclusion bodies, which were solubilized in guanidine-HCl. The protein was then purified by chromatography on immobilized cobalt, renatured by dilution, and further purified by anion exchange chromatography. Rabbit antiserum was raised against purified HEI, and this antiserum was used to generate polyclonal antibodies for Western blotting. A nitrocellulose membrane containing 5μg 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 NH₂-terminus sequence were indeed HEI. [0146] Figure IC again illustrates a nitrocellulose membrane containing 5μg ofthe MPR affinity-purified proteins, this time probed with radiolabeled MPR. Probing with this 2 nM ¹²⁵I-labeled soluble cation-independent MPR confirmed that HEI contained the Man6- P modification. Inclusion of 10 mM Man6-P during probing abrogated the signal. [0147] Northern blot analysis was performed to measure the level of HEI expression in various human tissues. 2 μg of polyadenylated RNA from various human tissues (Origene Technologies, Rockville, MD) was immobilized on a membrane and probed with a ³²P- 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 β-galactosidase activity, the open bars represent tripeptidyl peptidase activity, and the hatched bars represent β-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.

[0149] The heavy mitochondrial (M) fraction and the light mitochondrial (L) fraction from the differential centrifugation experiment were pooled for analysis by isopycnic centrifugation as described in (41). The density gradient for isopycnic centrifugation was made of either sucrose or Nycodenz. Figure 2B shows the results of these isopycnic centrifugation experiments using fractions pooled from control rats. The dashed line represents the sucrose gradient. The upper panel represents Western blot analysis of each sucrose gradient fraction using rabbit antibodies to HEI . The lower panel represents lysosomal protein activity. As with the differential centrifugation results, there was a clear codistribution of HEI and the three lysosomal enzymes, showing that they reside in the same compartment.

[0150] Differential centrifugation was then performed on liver homogenate taken from rats treated with Triton WR1339, a nonionic detergent that accumulates in lysosomes and induces a striking and specific shift of these organelles in sucrose density gradients (35). The heavy mitochondrial (M) and light mitochondrial (L) fractions were again subjected to isopycnic centrifugation as described in (41), with a gradient made of either sucrose or Nycodenz. Figure 2C shows the results of these isopycnic centrifugation experiments using fractions pooled from treated rats. The dashed line represents the sucrose gradient. 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 [0151] Western blots were performed to determine the level of HEI 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 μg) was separated by SDS- polyacrylamide gel electrophoresis. The blot was probed with rabbit antibodies to HEI and chemilummescence. As a control, the blot was reprobed with rabbit antibodies to cathepsin D (Calbiochem, LaJolla, CA) and chemilummescence. 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, and lanes 10 and 11 represent NP-C2 disease samples. As seen in the top panel, HEI expression was undetectable in fibroblasts from the NP-C2 patients. In contrast, 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 ΝP-C2 patients [0152] 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. In this schematic, 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.

[0153] 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.

V. Correction of cholesterol accumulation in NP-C2 fibroblasts

[0154] The finding that P-C2 is caused by a deficiency in the lysosomal HEI protein led to experiments in which HEI protein was added to NP-C2 fibroblasts in trans to reverse the cholesterol accumulation phenotype.

[0155] A fragment corresponding to human HEI cDNA flanked by JKhol sites was generated using standard PCR-based methods and subcloned into -ϊbøl-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. [0156] Chinese hamster ovary (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 LIPOFECTAMINE™ (Gibco). Cells were selected with G418 and resistant clones isolated using cloning cylinders. A resistant clone that secreted mannose 6-phosphorylated HEI was subjected to multiple cycles of selection with increasing concentrations of MTX. Cells resistant to 400 μM MTX were subcloned and the clone that secreted the highest level of HEI was chosen for subsequent analysis. 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.

[0157] 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%> CO₂. 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, while 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. As these results show, 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). 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. Addition of Man6- P to the HEI -conditioned media prevented the reduction in cholesterol accumulation, indicating that uptake occurred through MPR-mediated endocytosis. In contrast to the results obtained with NP-C2 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. [0158] For quantitation, 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).

[0159] The finding that 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).

[0160] As stated above, the foregoing is intended to be illustrative ofthe embodiments of the present invention, and are not intended to limit the invention in any way. Although the invention has been described with respect to the specific modifications described above, the details thereof are not to be construed as limitations, for it will be apparent that various equivalents, changes, and modifications may be resorted to without departing from the spirit and scope thereof and it is understood that such equivalent embodiments are to be included herein. REFERENCES

1. Brady, et al. 1966. The metabolism of sphingomyelin. II. Evidence of an enzymatic deficiency in Niemann-Pick disease. Proc. Natl. Acad. Sci. U.S.A. 55:366-369.

2. Brady, R.O. 1978. The Metabolic Basis of Inherited Disease. 4^th ed. (Stanbury, J.B., Wyngaarden, J.B., and Fredrickson, D.S., eds.) pp.718-730, McGraw-Hill, New York.

3. Brady, R.O., Filling-Katz, M.R., Barton, N. W., and Pentchev, P.G. 1989. Niemann-Pick Disease Types C and D. Neurol. Clin. 7:75-88.

4. Nanier, M.T., and Suzuki, K. 1998. Recent advances in elucidating Νiemann- Pick C disease. Brain Pathol. 8:163-174.

5. Pentchev, P.G., Nanier, M.T., Suzuki, K., and Patterson, M.C. 1995. The Metabolic and Molecular Bases of Inherited Disease, 7^th ed. (Scriver, C.R., Beaudet, A.L., Sly, W.S., and Nalle, J.B., eds.) pp. 2625-2639, McGraw-Hill, New York.

6. Christmanou, H., et al. 2000. Deficient ferritin immunoreactivity in tissues from Niemann-Pick type C patients: extension of findings to fetal tissues, H and L ferritin isoforms, but also one case ofthe rare Niemann-Pick C2 complementation group. Mol. Genet. Metab. 70:196-202.

7. Alpy, F., et al. 2001. The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein. J. Biol. Chem. 276:4261- 4269.

8. Marx, J. 2001. Disease genes clarify cholesterol trafficking. Science 290:5500.

9. Nanier, M.T., Duthel, S., Rodriguez-Lafrasse, C, Pentchev, P., and Carstea,

E.D. 1996. Genetic heterogeneity in Νiemann-Pick C disease: a study using somatic cell hybridization and linkage analysis. Am. J. Hum. Genet. 58:118-125.

10. Steinberg, S.J., Ward, C.P., and Fensom, A.H. 1994. Complementation studies in Νiemann-Pick disease type C indicate the existence of a second group. J. Med. Genet. 31:317-320.

11. Steinberg, S.J., Mondal, D., and Fensom, A.H. 1996. Co-cultivation of Νiemann-Pick disease type C fibroblasts belonging to complementation groups alpha and beta stimulates LDL-derived cholesterol esterification. J. Inherit. Metab. Dis. 19:769-774. 12. Carstea, E.D., et al. 1997. Niemann-Pick Cl disease gene: homology to mediators of cholesterol homeostasis. Science 277:228-231.

13. Davies, J.P., Chen, F.W., and Ioannou, Y. A. 2000. Transmembrane molecular pump activity of Niemann-Pick Cl protein. Science 290:2295-2298.

14. Millat, G., et al. 2001. Niemann-pick disease type c: spectrum of hel mutations and genotype/phenotype correlations in the npc2 group. Am. J. Hum. Genet. 69:1013-1021.

15. Kirchhoff, C, Osterhoff, C, Habben, I., and Ivell, R. 1990. Cloning and analysis of mRNAs expressed specifically in the human epididymis. Int. J. Androl. 13:155-167.

16. KruU, N., Ivell, R., Osterhoff, C, Kirchhoff, C. 1993. Region-specific variation of gene expression in the human epididymis as revealed by in situ hybridization with tissue-specific cDNAs. Molec. Reprod. Dev. 34:16-24.

17. Kirchhoff, C, Osterhoff, C, Young, L. 1996. Molecular cloning and characterization of HEI, a major secretory protein ofthe human epididymis. Biol. Reprod. 54:847-856.

18. Fouchecourt, S., Metayer, S., Locatelli, A., Dacheux, F., and Dacheux, J.L.

2000. Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins. Biol. Reprod. 62:1790-1803.

19. Okamura, N., et al. 1999. A porcine homolog of the major secretory protein of human epididymis, HEI, specifically binds cholesterol. Biochim. Biophys. Acta 1438:377-387.

20. Frohlich, O., and Young, L.G. 1996. Molecular cloning and characterization of EPI-1, the major protein in chimpanzee (Pan troglodytes) cauda epididymal fluid. Biol. Reprod. 54:857-864.

21. Ellerbrock, K., Pera, I., Hartung, S., and Ivell, R. 1994. Gene expression in the dog epididymis: a model for human epididymal function. Int. J. Androl. 17:314-

323.

22. Larsen, L.B., Ravn, P., Boisen, A., Berglund, L., and Petersen, T.E. 1997. Primary structure of EPV20, a secretory glycoprotein containing a previously uncharacterized type of domain. Eur. J. Biochem. 243:437-441. 23. Nakamura, Y., et al. 2000. Primary structure, genomic organization and expression ofthe major secretory protein of murine epididymis, ME1. Gene 251:55-62.

24. Kirchhoff, C, Osterhoff, C, Pera, I., and Schroter, S. 1998. Function of human epididymal proteins in sperm maturation. Andrologia 30:225-232.

25. Naureckiene, S. 2000. Identification of HEI as the second gene of Niemann-Pick C disease. Science 290:2298-2301.

26. Brown, D., and Breton, S. 2000. H(+)V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways. J. Exp. Biol. 203:137-145.

27. Raczek, S., et al. 2000. Immunocytochemical localisation of some lysosomal hydrolases, their presence in luminal fluid and their directional secretion by human epididymal cells in culture. Cell Tissue Res. 280:415-425.

28. Tomomasa, H., et al. 1994. Lysosomal cysteine proteinases in rat epididymis. J. Histochem. Cytochem. 42:417-425.

29. Marx, J. 2001. Bad for the heart, bad for the mind?. Science 294:508-509.

30. Patterson, M.C., et al. 2001. The Metabolic and Molecular Bases of Inherited Disease, 8^th ed. (Scriver, C.R., et al, eds.) pp. 3611-3634, McGraw-Hill, New York.

31. Sleat, D.E., et al. 1997. Associations of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis. Science 277:1802-1805.

32. Sleat, D.E., Sohar, I., Lackland, H., Majercak, J., and Lobel, P. 1996. 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.

33. Kornfeld, S., and Sly, W.S. 2001. The Metabolic and Molecular Bases of Inherited Disease, 8^th ed. (Scriver, C.R., et al, eds.) pp. 3469-3482, McGraw-Hill, New York.

34. Nalenzano, K.J., Kallay, L.M., and Lobel, P. 1993. An assay to detect glycoproteins that contain mannose 6-phosphate. Anal. Biochem. 209:156-162.

35. Wattiaux, M., Wabo, P., and Baudhuin, P. 1963. Ciba Foundation Symposium "Lysosomes". pp. 176-200, Brown, Boston. 36. Nanier, M.T., et al. 1991. Type C Νiemann-Pick disease: spectrum of phenotypic variation in disruption of intracellular LDL-derived cholesterol processing. Biochim. Biophys. Acta 1096:328-337.

37. Higgins, M.E., Davies, J.P., Chen, F.W., and loannou, Y.A. 1999. Νiemann- Pick Cl is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network. Mol. Genet. Metab. 68:1-13.

38. Νeufeld, E.B., et al. 1999. The Νiemann-Pick Cl protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo. J. Biol. Chem. 274:9627-9635.

39. de Duve, C, et al. 1955. Tissue fractionation studies 6. Intracellular distribution patterns of enzymes in rat liver tissue. Biochem. J. 60:604-612.

40. Sohar, I., Lin, L, and Lobel, P. 2000. Enzyme-based diagnosis of classical late infantile neuronal ceroid lipofuscinosis: comparison of tripeptidyl peptidase I and pepstatin-insensitive protease assays. Clin. Chem. 46:1005-1008.

41. Jadot, M., Wattiaux-De Coninck, R., and Wattiaux, R. 1985. Effect on lysosomes of invertase endocytosed by rat-liver. Eur. J. Biochem. 151:485-488.

42. Νishiyama, C, Yuuki, T., Takai, T., Okumura, Y., and Okudaira, H. 1993. Determination of three disulfide bonds in a major house dust mite allergen, Der f II. Int. Arch. Allergy Immunol. 101:159-166.

43. Xiang, X., Han, G., Winkelmann, D. A., Zuo, W., and Morris, Ν.R. 2000. Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arpl. Curr. Biol. 10:603-106.

Claims

What is claimed is:

1. A method of diagnosing Niemann-Pick C2 disease in a subject, comprising detecting at least one mutation in the human HEI gene sequence of SEQ ID NO: 1.

2. The method of claim 1 wherein said mutation results in at least one mutation of an amino acid in the human HEI polypeptide sequence of SEQ ID NO: 2.

3. The method of claim 1 wherein said mutation is present in at least one of two copies of said HEI gene sequence, wherein said two copies are located on two separate chromosomes.

4. The method of claim 3 wherein said mutation indicates the ability of said subject to genetically transmit Niemann-Pick C2 disease.

5. The method of claim 3 wherein said mutation is present in both copies of said HEI gene sequence.

6. The method of claim 5 wherein said mutation indicates the Niemann-C2 disease in said subject.

7. The method of claim 5 wherein said mutation is homozygous.

8. The method of claim 5 wherein said mutation is heterozygous.

9. The method of claim 1 wherein said mutation is selected from the group consisting of: deletion, substitution, splice mutation, trimcation, frame-shift and insertion.

10. The method of claim 1 wherein said mutation is detected by the steps comprising:

(a) nucleic acid amplification; and

(b) subsequent sequence analysis ofthe nucleic acid.

11. The method of claim 10 wherein said amplification consist of PCR amplification.

12. The method of claim 11 wherein the nucleic acid is a human HEI gene.

13. The method of claim 11 wherein said mutation is detected by hybridizing:

(a) the HEI gene of said subject; and

(b) a labeled polynucleotide probe complementary to a portion ofthe wild type HEI nucleotide sequence of SEQ ID NO: 1.

14. The method of claim 13 wherein said polynucleotide probe is DNA.

15. The method of claim 13 wherein said label is selected from the group consisting of: a radioactive isotope, biotin, enzyme reporter group, chemiluminescent label, fluorescent label, and an antibody.

16. The method of claim 11 wherein said HEI gene of said subject is amplified by method selected from the group consisting of PCR and RT-PCR.

17. The method of claim 13 wherein the medium for said hybridization is selected from the group consisting of solid support, liquid solution, in situ, and Southern blot.

18. The method of claim 13 wherein said subject is a fetus.

19. A composition comprising a biologically effective amount of an HEI polynucleotide.

20. The composition of claim 19 further comprising an acceptable biological carrier.

21. The composition of claim 19 wherein said HEI polynucleotide sequence is the sequence of SEQ ID NO: 1.

22. The composition of claim 19 wherein said HEI polynucleotide sequence has at least 70%> identity to SEQ. ID. NO: 1, said identity being calculated over the entire length of SEQ. ID. NO: 1.

23. The composition of claim 19 further comprising a vector, which comprises a polynucleotide, wherein said polynucleotide encodes an HEI polypeptide.

24. The composition of claim 23 further comprising an expression system, which comprises:

(a) a cassette with an HEI gene,

(b) a vector; and

(c) a biologically acceptable carrier.

25. The expression system of claim 24 wherein said expression cassette contains an HEI nucleic acid element selected from the group consisting of HEI DNA, HEI cDNA, HEI antisense sequence, and HEI RNA.

26. The expression system of claim 25 wherein said expression cassette contains one or more elements selected from the group consisting ofthe following: (a) a promoter sequence;

(b) a transcription initiation sequence;

(c) one or more enhancer sequences;

(d) one or more selectable elements; and

(e) a reporter gene.

27. The expression system of claim 24 wherein said vector is selected from the group consisting of: a plasmid, cosmid, lambda phage, Ml 3 phage, a retrovirus, lentivirus, adenovirus, herpes simplex virus (HSV), cytomegalovims (CMV), adeno-associated virus (AAV), papillomavirus, and simian virus (SV40).

28. The expression system of claim 24 wherein said expression system is capable of expressing an HEI polypeptide substantially identical to SEQ ID NO: 2 in a host cell.

29. A composition comprising a biologically effective amount of an HEI polypeptide and an acceptable carrier.

30. The composition of claim 29 wherein said HEI polypeptide sequence is the sequence of SEQ ID NO: 2.

31. The composition of claim 29 wherein said HEI polypeptide sequence is substantially similar to SEQ. ID. NO: 2.

32. A method of altering intracellular cholesterol levels comprising administering an effective amount of an HEI therapeutic.

33. The composition of claim 32 further comprising an acceptable biological carrier.

34. The method of claim 32 wherein said HEI therapeutic is selected from the group consisting of: a HEI DNA polynucleotide sequence, a HEI RNA polynucleotide sequence, a HEI antisense polynucleotide sequence, a HEI polypeptide and a HEI protein.

35. The method of claim 32 wherein said levels are decreased by administration of a HEI therapeutic selected from the group consisting of: a HEI DNA polynucleotide sequence, a HEI RNA polynucleotide sequence, a HEI polypeptide and a HEI protein.

36. The method of claim 35 wherein said HEI therapeutic comprises an HEI polynucleotide sequence identical to SEQ ID NO: 1.

37. The method of claim 32 wherein said HEI therapeutic comprises an HEI polynucleotide antisense sequence complementary to SEQ ID NO: 1.

38. The method of claim 37 wherein cholesterol levels are increased by said administration of said HEI polynucleotide antisense sequence.

39. The method of claim 35, wherein said HEI therapeutic comprises an HEI protein sequence substantially similar to SEQ ID NO: 2.

40. The method of claim 32, wherein said administration comprises introducing into a target cell an effective amount of an HEI polynucleotide sequence.

41. The method of claim 40 wherein said introduction is performed by transfection.

42. The method of claim 41 wherein said introduction is carried out by a method selected from the group consisting of electroporation, microinjection, ballistic introduction or infection, transvection, calcium phosphate transfection, DAEA-dextran mediated transfection, scrape loading, use of a gene gun, cationic lipid-mediated transfection, and lipofection.

43. The method of claim 41 wherein said transfection occurs in vivo .

44. The method of claim 41 wherein said transfection occurs in vitro.

45. The method of claim 41 wherein said transfection takes place as part of an ex vivo procedure.

46. The method of claim 32 wherein said administration is to a human.

47. The method of claim 32 wherein said subject is a fetus.

48. The method of claims 46 and 47 wherein said administration comprises delivering said HEI therapeutic by a method selected from the group consisting of: intravascular injection, intramuscular injection, and oral administration.

49. The method of claim 32 wherein the alteration of intracellular cholesterol level is used to treat at least one disease selected from the group consisting of Niemann-Pick C2 disease, cardiovascular disease, atherosclerosis, Alzheimer's disease, and diabetes.

50. The method of claim 39 wherein said administration of HEI protein downregulates endogenous cholesterol synthesis.

51. A method of diagnosing Niemann-Pick Cl disease in a subject, comprising detecting elevated expression levels ofthe HEI gene in a cell.

52. The method of claim 51 wherein said elevated expression levels of HEI are detected by the steps comprising:

(a) measuring the amount of HEI mRNA in the cells of said subject; and

(b) comparing the results with the amount of HEI mRNA in the cells of a healthy subject.

53. The method of claim 52 wherein the method of measuring said amount of HEI mRNA is selected from the group consisting of Northern blot, RT- PCR, and ribonuclease protection assay.

54. The method of claim 51 wherein said elevated expression levels of HEI are detected by the steps comprising:

(a) measuring the amount of HEI protein in the cells of said subject; and

(b) comparing the results with the amount of HEI protein in the cells of a healthy subject.

55. The method of claim 54 wherein the method of measuring said amount of HEI protein is Western blotting.

56. The method of claim 51 wherein said subject is a human.

57. The method of claim 51 wherein the cell is a fibroblast.