METHODS AND COMPOSITIONS FOR GENE THERAPY FOR THE TREATMENT OF DEFECTS IN LIPOPROTEIN METABOLISM

Abstract: The invention provides a recombinant viral vector comprising the DNA of, or corresponding to, at least a portion of the genome of an adenovirus, which portion is capable of infecting a hepatic cell; and a human VLDL receptor gene operatively linked to regulatory sequences directing its expression. The vector is capable of expressing the normal VLDL receptor gene product in hepatic cells in vivo or in vitro. This viral vector is useful in the treatment of metabolic disorders caused by the accumulation of LDL in plasma, such as familial hypercholesterolemia or familial combined hyperlipidemia.

Description: METHODS AND COMPOSITIONS FOR GENE THERAPY FOR THE TREATMENT OF DEFECTS IN LIPOPROTEIN METABOLISM This invention was supported by the National Institute of Health Grant Nos. DK 42193-05 and HD 29946. The United States government has rights in this invention. Field of the Invention The present invention relates to the field of somatic gene therapy and the treatment of genetic disorders related to lipoprotein metabolism. Background of the Invention The metabolism of lipids, particularly cholesterol, involves the interaction of a number of lipoproteins and apolipoproteins. Very low density lipoprotein (VLDL) and apolipoprotein E (apoE) are key precursor molecules in the production of low density lipoprotein (LDL) and in the overall metabolism of lipids, including cholesterol. LDL is the major cholesterol-transport lipoprotein in human plasma. The VLDL IapoE receptors are expressed in heart, skeletal muscle, and adipose tissue [F. M. ittmaack et al, Endocrinol.. 136(1) ;340-348 (1995)] with lower levels of expression in the kidney, placenta, pancreas, and brain. This receptor has been suggested to play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs. The cDNA encoding the putative human VLDL receptor was recently cloned [M. E. Gafvels et al, Som. Cell Mol. Genet.. 19:557-569 (1993), incorporated by reference herein] . The receptor for LDL is located in coated pits on the surfaces of cells in the liver and other organs. As depicted in Fig. 1A, in a normal healthy human, the molecules apolipoprotein B48 (Apo-B48) , apolipoprotein C-II (Apo-C-II) and Apo E form a chylo icron particle in plasma passing through the intestines, which interacts with a chylomicron remnant receptor in the liver. After metabolism of the chylo icrons taken up by the remnant receptor, the liver produces the primary lipoprotein, VLDL, which contains Apo-E, Apo-C-II and apolipoprotein B100 (Apo B100) . VLDL is metabolized into LDL, which binds to the LDL receptor in the liver via Apo B100. The LDL receptor in the liver facilitates the uptake of LDL by receptor-mediated endocytosis. LDL is degraded in lysoso es, and its cholesterol is released for metabolic use. Defects in the metabolism of such lipoproteins and/or receptors result in several serious metabolic disorders. The human disease familial hyper- cholesterolemia (FH) is caused primarily by one or more mutations in the gene encoding the LDL receptor. FH is characterized clinically by (1) an elevated concentration of LDL; (2) deposition of D -derived cholesterol in tendons and skin (xanthomas) and in arteries (atheromas) ; and (3) inheritance as an autosomal dominant trait with a gene dosage effect. Individuals with FH develop premature coronary heart disease, usually in childhood. Heterozygotes number about 1 in 500 persons, placing FH among the most common inborn errors of metabolism. Heterozygotes have twofold elevations in plasma cholesterol (350 to 550 mg/dl) from birth and tend to develop tendon xanthomas and coronary atherosclerosis after age 20. Homozygotes number 1 in 1 million persons and are characterized by severe hypercholesterolemia (650 to 1000 mg/dl) , cutaneous xanthomas which appear within the first 4 years of life, and coronary heart disease which begins in childhood and frequently causes death before age 20. [J. Goldstein et al, "Familial Hypercholesterolemia 1 ', Chapter 48, in The Metabolic Basis of Inherited Disease. 6th ed. , C. R. Scrivers et al (eds) , McGraw-Hill Information Services Co. , NY, NY, (1989) pp. 1215-1250]. Another metabolic disorder is familial combined hyperlipidemia (FCH) which was first associated with hyperlipidemia in survivors of myocardial infarction and their relatives. FCH patients generally have one of three phenotypes: (1) elevated levels of VLDL, (2 ) elevated levels of LDL, or (3) increases in the levels of both lipoproteins in plasma. Unlike FH, FCH appears in only 10 to 20 percent of patients in childhood, usually in the form of hypertriglyceridemia. Homozygosity for the trait may result in severe hypertriglyceridemia. [J. Goldstein et al, "Disorders of the Biogenesis and Secretion of Lipoproteins", Chapter 44B in The Metabolic Basis of Inherited Disease. 6th ed., C. R. Scrivers et al (eds), McGraw-Hill Information Services Co., NY, NY, (1989) pp. 1155-1156]. This disorder is also associated with the appearance of glucose intolerance and obesity in a number of individuals. The most striking abnormality of FCH is marked elevation of VLDL content of plasma. Increased production of VLDL leads to an expanded plasma pool of VLDL in some individuals, but in others with more efficient lipolysis, it results in increased levels of LDL. FCH is characterized by an excess production of LDL, rather than a genetic defect in the LDL receptor. The LDL receptors of cultured fibroblasts appear to be normal in FCH patients. Clinical experience suggests that FCH is at least five times as prevalent as FH, occurring in about 1 percent of the North American population. The predilection toward coronary artery disease among patients with this disorder makes it the most prominent known metabolic cause of premature atherosclerosis [J. Goldstein et al, cited above]. When LDL receptors are deficient as in FH (see Fig. IB) , or excess LDL is produced due to excess VLDL as in FCH, the efficient removal of LDL from plasma by the liver declines, and the level of LDL rises in inverse proportion to the receptor number. The excess plasma LDL is deposited in connective tissues and in scavenger cells, resulting in the symptoms of either disorder. Presently, treatment for FH and FCH is directed at lowering the plasma level of LDL by the administration of drugs, i.e., combined administration of a bile acid- binding resin and an inhibitor of 3-hydroxy-3- methylglutaryl CoA reductase for treatment of FH and niacin for treatment of FCH. However, FH homozygotes with two nonfunctional genes are resistant to drugs that work by stimulating LDL receptors, similarly, such drugs are not particularly effective in FCH. In FH homozygotes, plasma LDL levels can be lowered only by physical or surgical means. Administration of normal LDL receptor genes by gene therapy using an adenovirus vector has been contemplated for the treatment of FH. Adenovirus vectors are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state. The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders [K. F. Kozarsky et al, Somatic Cell Mol. Genet.. 19:449-458 (1993) ("Kozarsky I") ; K. F. Kozarsky et al, J. Biol. Che .. 2jS9:l3695- 13702 (1994) ("Kozarsky II); Y. Watanabe, Atherosclerosis. 2£:261-268 (1986); K. Tanzawa et al, FEBS Letters. H8(l):81-84 (1980); J.L. Golasten et al, New Enαl. J. Med.. 301:288-296 (1983); S. Ishibashi et al, J. Clin. Invest.. £2:883-893 (1993); and S. Ishibashi et al, J. Clin. Invest.. 92:1885-1893 (1994)]. The use of adenovirus vectors in the transduction of genes into hepatocytes in vivo has previously been demonstrated in rodents and rabbits [see, e.g., Kozarsky II, cited above, and S. Ishibashi et al, J. Clin. Invest.. 92:883-893 (1993)]. Recent research has shown that introduction of a recombinant adenovirus encoding the human LDL receptor ("LDLR") cDNA into the livers of LDL receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbits, which mimic the condition of FH, resulted in large, transient reductions in plasma cholesterol. The transient nature of the effect of recombinant adenoviruses in most situations is attributed to the development of cellular immune responses to the virus-infected cells and their subsequent elimination. Antigenic targets for immune mediated clearance are viral proteins expressed from the recombinant viral genome and/or the product of the transgene, which in this case, is the LDL receptor protein [Y. Yang et al, Proc. Natl. Acad. Sci.. USA. £1:4407-4411 (May 1994); Y. Yang et al, Immun.. 1:433-442 (August 1994) ] . Additionally, repeated reinfusions of the LDLR gene-containing adenovirus did not produce similar, subsequent cholesterol reductions due to the development of neutralizing anti-adenovirus antibodies [Kozarsky I and Kozarsky II, cited above; see also Y. Yang et al, Immun. , 1:433-442 (August 1994), all incorporated by reference herein] . There remains a need in the art for therapeutic compositions and gene therapy strategies which enable effective treatment and/or prevention of FH and FCH, as well as other defects in lipoprotein metabolism. smronarv of the invention In one aspect, the invention provides a recombinant viral vector comprising the DNA of, or corresponding to, at least a portion of the genome of an adenovirus, which portion is capable of infecting a hepatic cell; and a human VLDL receptor ( n VLDLR ) gene operatively linked to regulatory sequences directing its expression, the vector capable of expressing the VLDLR gene product in the hepatic cell in vivo or in vitro . In another aspect, the invention provides a mammalian cell infected with the viral vector described above. In still a further aspect, the invention provides a method for delivering and stably integrating a VLDLR gene into the chromosome of a mammalian hepatocyte cell comprising introducing into said cell an effective amount of a recombinant viral vector described above. Another aspect of this invention is a method for treating a patient having a metabolic disorder comprising administering to the patient by an appropriate route an effective amount of an above described vector containing a normal VLDLR gene, wherein said VLDLR gene is integrated into the chromosome of said patient's hepatocytes and said receptor is expressed stably in vivo at a location in the body where it is not normally expressed. Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof. Brief Description of the Drawings Fig. 1A is a schematic drawing of normal human and rabbit lipoprotein metabolism. The apolipoproteins are referred to as B48, B100, C-II, and E. LDL and VLDL are identified. Fig. IB is a schematic drawing of lipoprotein metabolism in FH patients and HHL rabbits. The abbreviations are as described in Fig. 1A. Fig. 1C is a schematic drawing of lipoprotein metabolism in rabbits infused with the recombinant VLDLR gene according to the invention. Fig. 2 is a schematic drawing of plasmid pAd.CMWI.DLR, which contains adenovirus map units 0-1 (Ad 0-1) , followed by a cytomegalovirus enhancer/promoter (CMV enh/prom) , a human VLDLR gene, a polyadenylation signal (pA) , adenovirus map units 9-16 (Ad 9-16) and plasmid sequences from plasmid pAT153 including an origin of replication and a picillin resistance gene. Restriction endonuclease enzymes are represented by conventional designations in the plasmid construct. Fig. 3 is a schematic map of recombinant adenovirus H5.010CMWLDLR, in which 0 to 100 represent the map units of an adenovirus type 5 (Genbank Accession No. M73260) , and the CMV/VLDLR/pA minicassette of pAd.CMWLDLR is inserted between adenovirus map units l and 9, with the remaining Ad5 map units 9-100 having a partial E3 gene deletion between about map unit 78.5 and about 84.3. Fig. 4A is a graph plotting changes in plasma cholesterol levels in mg/dl for WHHL rabbits as a function of days before and after receiving recombinant adenovirus H5.010CMVlacZ. The symbols represent individual animals. See Example 3. Fig. 4B is a graph plotting changes in plasma cholesterol levels in mg/dl for WHHL rabbits as a function of days before and after receiving recombinant adenovirus H5.010CMWLDLR. The symbols represent the response of four individual animals. See Example 3. Fig. 5 is a bar graph representing cholesterol levels (measured as % pre-infusion) in mice infused with recombinant adenovirus H5.OlOCMVlacZ (lacZ) , recombinant adenovirus H5.010CMWLDLR and recombinant adenovirus H5.010CBhLDLR. The dotted bars represent pre-infusion levels and the solid bars represent post-infusion levels. See Example 4. Fig. 6 is a bar graph representing cholesterol levels, specifically the levels of the fractions of plasma lipoproteins (measured as mg/fraction) in mice infused with recombinant adenovirus H5.OlOCMVlacZ (lacZ) , recombinant adenovirus H5.010CMWLDLR and recombinant adenovirus H5.010CBhLDLR. The solid bars represent proteins or fragments falling within a density (d) > 1.21; the thickly cross-hatched bars represent HDL; the closely cross-hatched bars represent LDL, the spaced apart slanted hatched bars represent intermediate density lipoprotein (IDL) , and the clear bars represent VLDL levels. See Example 4. Fig. 7A is a graph plotting changes in cholesterol levels (measured in mg/dl) as a function of days pre- and post-infusion for mice infused with H5.OlOCMVlacZ. The symbols represent the responses of individual animals. See Example 5. Fig. 7B is a graph plotting changes in cholesterol levels (measured in mg/dl) as a function of days pre- and post-infusion for mice infused with H5.010CBhLDLR. The symbols are the same as for Fig. 7A. See Example 5. Fig. 7C is a graph plotting changes in cholesterol levels (measured in mg/dl) vs. days pre and post-infusion for mice infused with H5.010CMWLDLR. The symbols are the same as for Fig. 7A. See Example 5. Fig. 7D is a graph providing the average results ± standard deviation from two experiments for mice infused with H5.010CMVLacZ (n=9) or with H5.010CMWLDLJ? (n=10) . Average pre-infusion cholesterol levels were 870 mg/dl and 946 mg/dl, respectively. Asterisks indicate p < 0.05. Figs. 8A-8F are the DNA sequence [SEQ ID NO: 1] with encoded a ino acid sequence [SEQ ID NO: 2] of the human VLDL receptor gene, as reported by Gafvels et al, cited above. Figs. 9A-9I are the DNA sequence of pAd.CMWLDLR [SEQ ID NO: 3], in which Ad 0-1 spans nucleotides 12-364, CMV ehn/prom spans nucleotides 381- 862; nucleotides 966-4107 encode VLDLR, pA spans nucleotides 4192-4390; Ad 9.2-16.1 span nucleotides 4417- 6880 and nucleotides 6881-9592 are pAT153 sequences. Fig. 10A is a bar chart illustrating the CTL activity (average ± standard deviation) measured at an effector:target cell ratio of 25:1. ** = p < 0.005; * = p < 0.05. Fig. 10B is a line graph illustrating the CTL activity measured against varying effector:target ratios. Fig. 11A is a graph summarizing neutralizing antibody titer present in BAL samples of C57BL/6 mice adenovirus-infected on day 0 and necrotized on day 28 as described in Example 9. Control represents normal mice ("control") ; CD4 mAB represents CD4 + cell depleted mice; IL-12 represents IL-12 treated mice and IFN-7 represent IFN-γ treated mice. Fig. 11B is a graph summarizing the relative amounts (OD 405 ) of IgG present in BAL samples. The symbols are as described in Fig. 11A. Fig. 11C is a graph summarizing the relative amounts (OD 4Q5 ) of IgA present in BAL samples. The symbols are as described in Fig. 11A. Detailed Description of the Invention The present invention provides novel compositions and methods which enable the therapeutic treatment of metabolic disorders, such as FH and FCH, characterized by the accumulation of LDL in human plasma. This invention provides for the use of a viral vector to introduce and stably express a gene normally expressed in mammals, i.e., the gene encoding a normal receptor for very low density lipoprotein (VLDLR) , in a location in the body where that gene is not naturally present, i.e., in the liver. The methods and compositions of the present invention overcome the problems previously identified in the gene therapy treatment of LDL receptor-deficient individuals. As described in detail below, by use of a viral vector capable of targeting cells of the liver, the VLDL receptor gene is introduced into and stably expressed in liver cells. The present invention differs from direct gene replacement in that the VLDL receptor protein is expressed normally in LDL receptor deficient individuals, e.g., the macrophages. Thus, gene therapy using a liver-directed viral vector carrying a VLDLR gene would result not in expression of a new gene product, but rather, in de novo expression in an organ which otherwise does not express the gene product. Importantly, the patient does not mount an immune response against the VLDLR gene product expressed in the liver because the vector-delivered VLDLR gene is not recognized as a foreign antigen, and there is no induction of CTL- mediated elimination of the transfected cell. In contrast, CTL-mediated elimination of viral vectors is a problem when an LDLR gene is administered to an LDLR- deficient individual with FH [see, e.g., Kozarsky I and II, cited above]. Due to this recognition of the VLDLR gene by the patient's immune system as a known gene, and to the tendency of hepatocytes to have a long life in circulation, the hepatocytes transfected with the vector of this invention, which express the VLDLR gene, tend to be stable and VLDLR expression is not transient. VLDLR gene expression in transfected hepatocytes occurs for the duration of the hepatocyte's life. The lipoprotein metabolic disorder may be treated for longer times without the need for reinfusing the viral vector, thus limiting the number of viral exposures and potential immune reactions to vector-encoded viral proteins. The vectors and methods of this invention can provide gene therapy useful to treat and/or supplement current treatments for lipoprotein metabolic disorders. The presence of the VLDL receptor gene in the transfected hepatocytes according to this invention permits the binding of VLDL, a precursor of LDL, from the plasma at the site of the liver, thereby decreasing the amount of VLDL in plasma. The decrease in VLDL in the plasma consequently decreases the production of plasma LDL . For example, in FH, this reduction in plasma LDL can compensate for the defective LDL receptors in the liver. In FCH, this reduced production of plasma LDL from VLDL prevents the normal LDL receptors in the liver from becoming overloaded by excess LDL, and reduces the excess VLDL which contributes to the disorder. Compare, for example, the schematic representations of the normal operation of lipid metabolism (Fig. 1A) to the abnormal metabolism caused by FH (Fig. IB) and then to the method of this invention (Fig. 1C) . J. Recombinant Viral Particles as Gene Therapy Vectors The compositions of this invention involve the construction of desirable gene therapy vectors, which are capable of delivering and stably integrating a functional, normal VLDL receptor gene to hepatocytes. Such gene therapy vectors include a selected virus vector, desirably deleted in one or more viral genes, a minigene containing the VLDLR gene under the control of regulatory sequences, and optional helper viruses and/or packaging cell lines which supply to the viral vectors any necessary products of deleted viral genes. The viral sequences used in the vectors, helper viruses, if needed, and recombinant viral particles, and other vector components and sequences employed in the construction of the vectors described herein are obtained from commercial or academic sources based on previously published and described sequences. These viral materials may also be obtained from an individual patient. The viral sequences and vector components may be generated by resort to the teachings and references contained herein, coupled with standard recombinant molecular cloning techniques known and practiced by those skilled in the art. Modifications of existing nucleic acid sequences forming the vectors, including sequence deletions, insertions, and other mutations taught by this specification may be generated using standard techniques. The methods employed for the selection of viral sequences useful in a vector, the cloning and construction of VLDLR "minigene" and its insertion into a desired viral vector and the production of a recombinant infectious viral particle by use of helper viruses and the like are within the skill in the art given the teachings provided herein. A. Construction of the "Minigene" By "minigene" is meant the combination of the VLDLR gene and the other regulatory elements necessary to transcribe the gene and express the gene product in vivo. The human VLDL receptor sequence has been provided [see, Gafvels et al, cited above; SEQ ID NOS: 1 and 2]. Generally, the entire coding region of this receptor sequence is used in the minigene; the 5' and 3 ' untranslated sequences of SEQ ID NO: 1 are not essential to the minigene. VLDL receptor genes of other mammalian origins, e.g., rabbit, monkey, etc., may also be useful in this invention. The VLDL receptor gene (VLDLR) is operatively linked to regulatory components in a manner which permits its transcription. Such components include conventional regulatory elements necessary to drive expression of the VLDLR transgene in a cell transfected with the viral vector. Thus the minigene also contains a selected promoter which is linked to the transgene and located, with other regulatory elements, within the selected viral sequences of the recombinant vector. Selection of the promoter is a routine matter and is not a limitation of this invention. Useful promoters may be constitutive promoters or regulated (inducible) promoters, which will enable control of the amount of the transgene to be expressed. For example, a desirable promoter is that of the cytomegalovirus immediate early promoter/enhancer [see, e.g., Boshart et al, Cell. 41:521-530 (1985)]. Another desirable promoter includes the Rous sarcoma virus LTR promoter/enhancer. Still another promoter/enhancer sequence is the chicken cytoplasmic β-actin promoter [T. A. Kost et al, Nucl. Acids Res.. 11(23) :8287 (1983)]. Other suitable promoters may be selected by one of skill in the art. The minigene may also desirably contain nucleic acid sequences heterologous to the viral vector sequences including sequences providing signals required for efficient polyadenylation of the transcript (poly-A or pA) and introns with functional splice donor and acceptor sites. A common poly-A sequence which is employed in the exemplary vectors of this invention is that derived from the papovavirus SV-40. The poly-A sequence generally is inserted in the minigene following the transgene sequences and before the viral vector sequences. A common intron sequence is also derived from SV-40, and is referred to as the SV-40 T intron sequence. A minigene of the present invention may also contain such an intron, desirably located between the promoter/enhancer sequence and the transgene. Selection of these and other common vector elements are conventional [see, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual.", 2d ed. , Cold Spring Harbor Laboratory, New York (1989) and references cited therein] and many such sequences are available from commercial and industrial sources as well as from Genbank. As stated above, the minigene is located in the site of any selected deletion in the viral vector. See Example 1 below. B . Construction of The Viral Plasmid Vector Although a number of viral vectors have been suggested for gene therapy, the most desirable vector for this purpose is a recombinant adenoviral vector or adeno-associated vector. Adenovirus vectors as described below are preferred because they can be purified in large quantities and highly concentrated, and the virus can transduce genes into non-dividing cells. However, it is within the skill of the art for other adenovirus, or even retrovirus, vaccinia or other virus vectors to be similarly constructed. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types. Human adenoviruses comprise a linear, approximately 36 kb double-stranded DNA genome, which is divided into 100 map units (m.u.), each of which is 360 bp in length. The DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication. The gene products are organized into early (El through E4) and late (LI through L5) regions, based on expression before or after the initiation of viral DNA synthesis [see, e.g., Horwitz, Virology. 2d edit., ed. B. N. Fields, Raven Press, Ltd. , New York (1990)]. The general adenoviruses types 2 and 5 (Ad2 and Ad5, respectively) , are not associated with human malignancies. Suitable adenovirus vectors useful in gene therapy are well known [see, e.g., M. S. Horwitz et al, "Adenoviridae and Their Replication", Virology, second edition, pp. 1712, ed. B. N. Fields et al, Raven Press Ltd., New York (1990); M. Rosenfeld et al, Cell. 68:143- 155 (1992); J. F. Engelhardt et al, Human Genet. Ther.. 4.:759-769 (1993); Y. Yang et al, Nature Genet.. 2:362-269 (1994); J. Wilson, Nature. 36^:691-692 (Oct. 1993); B. J. Carter, in "Handbook of Parvoviruses", ed. P. Tijsser, CRC Press, pp. 155-168 (1990). The selection of the adenovirus type is not anticipated to limit the following invention. Adenovirus vectors useful in this invention may include the DNA sequences of a number of adenovirus types. The adenovirus sequences useful in the vectors described herein may be obtained from any known adenovirus type, including the presently identified 41 human types [see, e.g., Horwitz, cited above]. The sequence of a strain of adenovirus type 5 may be readily obtained from Genbank Accession No. M73260. Similarly, adenoviruses known to infect other animals may also be employed in the vector constructs of this invention. A variety of adenovirus strains are available from the American Type Culture Collection, Rockville, Maryland, or available by request from a variety of commercial and institutional sources. Adenovirus vectors useful in this invention include recombinant, defective adenoviruses, optionally bearing other mutations, e.g. , temperature- sensitive mutations, deletions and hybrid vectors formed with adenovirus/adeno-associated virus sequences. Suitable vectors are described in the published literature [see, for example, Kozarsky I and II, cited above, and references cited therein, U. S. Patent No. 5,240,846 and the co-pending applications incorporated herein by reference below. Useful adenovirus vectors for delivery of the VLDLR gene to the liver, minimal adenovirus nucleic acid sequences may be used to make a vector, in which case the use of a helper virus to produce a hybrid virus particle is required. Alternatively, only selected deletions of one or more adenovirus genes may be employed to construct a viral vector. Deleted gene products can be supplied by using a selected packaging cell line which supplies the missing gene product. 1. Recombinant Minimal Adenovirus Desirable adenovirus (Ad) vectors useful in the present invention are described in detail in co-pending, co-owned U.S. Patent Application Serial No. 08/331,381, which is incorporated by reference herein for the purpose of describing these vectors. Briefly summarized, the minimal Ad virus is a viral particle containing only the adenovirus cis-elements necessary for replication and virion encapsidation, but otherwise deleted of all adenovirus genes. That is, the vector contains only the cis-acting 5' and 3' inverted terminal repeat (ITR) sequences of an adenovirus (which function as origins of replication) and the native 5' packaging/enhancer domain, that contains sequences necessary for packaging linear Ad genomes and enhancer elements for the El promoter. This left terminal (5') sequence of the Ad5 genome spans bp 1 to about 360 of the conventional published Ad5 adenovirus genome, also referred to as map units 0-1 of the viral genome, and generally is from about 353 to about 360 nucleotides in length. This sequence includes the 5'ITR (bp 1 to about 103 of the adenovirus genome) ; and the packaging/enhancer domain (bp about 194 to about 358 of the adenovirus genome) . The minimal 3 • adenovirus sequences of the adenovirus vector may include the right terminal (3' ITR sequence of the adenoviral genome spanning about bp 35,353 to the end of the adenovirus genome, or map units -98.4-100. This sequence is generally about 580 nucleotide in length. Between such sequences, a VLDLR minigene, as described above, is inserted. Production of an infectious particle from this minimal Ad viral vector involves the assistance of a helper virus, as discussed below. A second type of minimal vector also disclosed in the above-incorporated reference places the 5' Ad terminal sequence in a head- to-tail arrangement relative to the 3' terminal sequence. The minimal Ad vector co-infected with a helper virus and/or a packaging cell line provides all of the viral gene products necessary to produce an infective recombinant viral particle containing the VLDLR minigene. Alternatively, this vector can contain additional adenovirus gene sequences, which then are not required to be supplied by a helper virus. 2. Other Defective Adenoviruses Recombinant, replication-deficient adenoviruses useful for gene therapy of this invention may be characterized by containing more than the minimal adenovirus sequences defined above. These other Ad vectors can be characterized by deletions of various portions of gene regions of the virus, and infectious virus particles formed by the optional use of helper viruses and/or packaging cell lines. Suitable defective adenoviruses are described in more detail in Kozarsky and Wilson, Curr. Opin. Genet. Devel.. 2:499-503 (1993); Kozarsky I and II, cited above, and references cited therein, all incorporated herein by reference. As one example, suitable vectors may be formed by deleting all or a sufficient portion of the adenoviral early immediate early gene Ela (which spans mu 1.3 to 4.5) and delayed early gene Elb (which spans mu 4.6 to 11.2) so as to eliminate their normal biological functions. These replication-defective El-deleted viruses are capable of replicating and producing infectious virus when grown on an adenovirus-transformed, complementation human embryonic kidney cell line, the 293 cell [ATCC CRL1573], containing functional adenovirus Ela and Elb genes which provide the corresponding gene products in trans. The resulting virus is capable of infecting many cell types and can express a transgene (i.e., VLDLR gene), but cannot replicate in most cells that do not carry the El region DNA unless the cell is infected at a very high multiplicity of infection. Extensive experience in animals indicates that El-deleted vectors are not particularly desirable for gene therapy because low levels of viral proteins are expressed which elicit destructive cellular immune responses. As a preferred example, all or a portion of the adenovirus delayed early gene E3 (which spans mu 76.6 to 86.2) may be eliminated from the adenovirus sequence which forms a part of the hybrid construct. The function of E3 is irrelevant to the function and production of the recombinant virus particle. For example, Ad vectors may be constructed with a therapeutic minigene inserted into the El-deleted region of the known mutant Ad5 sub360 backbone [J. Logan et al, Proc. Natl. Acad. Sci. USA. £1:3655-3659 (1984)]; or the Ad5 mutant dl7001 backbone [Dr. William Wold, Washington University, St. Louis]. Both mutant viruses also contain a deletion in the E3 region of the adenoviral genome; in sub360, at 78.5 to 84.3 mu, and in dl7001, at 78.4 to 86 mu. The life cycle of both sub360 and dl7001 display wild type characteristics. More preferred adenovirus vectors may be constructed having a deletion of the El gene, at least a portion of the E3 region, and an additional deletion within adenovirus genes other than El and E3 to accommodate the VLDLR minigene and/or other mutations which result in reduced expression of adenoviral protein and/or reduced viral replication. For example, all or a portion of the adenovirus delayed early gene E2a (which spans mu 67.9 to 61.5) may be eliminated from the adenovirus vector. It is also anticipated that portions of the other delayed early genes E2b (which spans mu 29 to 14.2) and E4 (which spans mu 96.8 to 91.3) may also be eliminated from the adenovirus vector. Deletions may also be made in any of the late genes LI through L5, which span mu 16.45 to 99 of the adenovirus genome. Similarly, deletions may be useful in the intermediate genes IX (which maps between u 9.8 and 11.2) and IVa 2 (which maps between 16.1 to 11.1). Other useful deletions may also be made in the other structural or non-structural adenovirus genes. An adenovirus sequence for use in the present invention may contain deletions of El only. Alternatively, deletions of entire genes or portions effective to destroy their biological activity may be used in any combination. For example, in one exemplary vector, the adenovirus sequence may contain deletions of the El genes and the E3 gene, or of the El, E2a and E3 genes, or of the El and E4 genes, or of El, E2a and E4 genes, with or without deletion of E3, and so on. Vectors may also contain additional mutations in genes necessary for viral replication. Adenovirus vectors may contain a mutation which produces temperature-sensitive (ts) viruses. Among such mutations include the incorporation of the missense temperature- sensitive mutation in the E2a region found in the Ad5 H5tsl25 strain [P. Vander Vliet et al, J. Virol.. 15:348- 354 (1975)] at 62.5 u. A single amino acid substitution (62.5 mu) at the carboxy end of the 72 kd protein (DBP) produced from the E2a gene in this strain produces a protein product which is a single-stranded DNA binding protein and is involved in the replication of adenoviral genomic DNA. At permissive temperatures (approximately 32°C) the ts strain is capable of full life cycle growth on HeLa cells, while at non-permissive temperatures (approximately 38°C) , no replication of adenoviral DNA is seen. In addition, at non-permissive temperatures, decreased immunoreactive 72 kd protein is seen in HeLa cells. Exemplary vectors for use in this invention, for example, may be obtained by combining fragments from three independent DNA constructs, including sub360 or dl7001, H5tsl25, and a cDNA plasmid with Ela sequences placed 5' to a therapeutic minigene. This type of vector is described, for example, by J. F. Engelhardt et al, Proc. Natl. Acad. Sci. USA. 91:6196- 6200 (June 1994); Y. Yang et al, Nature Genet.. 7: 362- 369 (July, 1994) and references cited therein, all references incorporated herein by reference. Due to the mutations in the vector, there is reduced viral replication, reduction in expressed protein and an increase in the persistence of transgene expression. Other preferred adenovirus vectors contain the H5tsl25 mutation in addition to E3 deletions of sub360 and dl7001. The minigene containing VLDLR as the transgene may be inserted into any deleted region of the selected Ad virus. An exemplary Ad virus vector used to demonstrate this invention is the defective adenovirus vector H5.010CMWLDLR, which contains adenovirus sequences Ad m.u. 0-1, followed by a VLDLR minigene, and the sequence Ad m.u.9 to 100 with small deletions in E3. See Fig. 3, described above. The recombinant adenovirus was fully deleted of Ela, Elb and partially deleted of E3. This recombinant virus vector is described in detail in Example 1. 3. Ad/AAV Hybrid Vectors Another preferred vector is a hybrid Ad/AAV vector, which is the subject of co-owned, co- pending U.S. Patent Application Ser. No. 08/331,384, which is incorporated by reference herein. At a minimum, the adenovirus nucleic acid sequences employed in the hybrid vector of this invention are the minimal adenovirus geno ic sequences required for packaging adenoviral genomic DNA into a preformed capsid head, as described above. The entire adenovirus 5' sequence containing the 5'ITR and packaging/enhancer region can be employed as the 5' adenovirus sequence in the hybrid vector. The 3' adenovirus sequences of the vector include the right terminal (3') ITR sequence of the adenoviral genome discussed above. Some modifications to these sequences which do not adversely affect their biological function may be acceptable. Also part of the hybrid vectors of this invention are sequences of an adeno-associated virus. The AAV sequences useful in the hybrid vector are the viral sequences from which the rep and cap polypeptide encoding sequences are deleted. More specifically, the AAV sequences employed are the cis- acting 5' and 3' inverted terminal repeat (ITR) sequences [See, e.g., B. J. Carter, cited above]. The AAV ITR sequences are about 143 bp in length. Substantially the entire sequences encoding the ITRs are used in the vectors, although some degree of minor modification of these sequences is expected to be permissible for this use. The ability to modify these ITR sequences is within the skill of the art. See, e.g., Sambrook et al, cited above. In the Ad/AAV hybrid vector construct, the AAV sequences are flanked by the adenovirus sequences discussed above. The 5' and 3' AAV ITR sequences themselves flank a VLDLR minigene sequence as described above. Thus, the sequence formed by the VLDLR minigene and flanking 5' and 3' AAV sequences may be inserted at any deletion site in the adenovirus sequences of the vector. For example, the AAV sequences are desirably inserted at the site of deleted Ela/Elb genes of the adenovirus, i.e., after map unit 1. Alternatively, the AAV sequences may be inserted at an E3 deletion, E2a deletion, and so on. If only the adenovirus 5• ITR/packaging sequences and 3• ITR sequences are used in the vector, the AAV sequences are inserted between them. As described above for the minimum adenovirus sequences, those gene sequences not present in the adenovirus portion of the hybrid vector must be supplied by either a packaging cell line and/or a helper adenovirus to generate the recombinant hybrid viral particle. Uptake of this hybrid virus by the cell is caused by the infective ability contributed to the vector by the adenovirus and AAV sequences. Once the virus or virus conjugate is taken up by a cell, the AAV ITR flanked transgene must be rescued from the parental adenovirus backbone. Rescue of the transgene is dependent upon supplying the infected cell with an AAV rep gene. The AAV rep gene can be supplied to the hybrid virus by several methods described in the above-incorporated application. One embodiment for providing rep proteins in trans is by transfecting into the target monolayer of cells previously infected with the hybrid vector, a liposome enveloped plasmid containing the genes encoding the AAV rep 78 kDa and 52 kDa proteins under the control of the AAV P5 promoter. More preferably for in vivo use, the AAV rep gene may also be delivered as part of the hybrid virus. One embodiment of this single particle concept is supplied by a polycation conjugate of hybrid virus. Infection of this modified virus conjugate is accomplished in the same manner and with regard to the same target cells as identified above. However, the polylysine conjugate of the hybrid virus onto which was directly co plexed a plasmid that encoded the rep 78 and 52 proteins, combines all of the functional components into a single particle structure. Thus, the hybrid virus conjugate permits delivery of a single particle to the cell, which is considerably more desirable for therapeutic use. In another embodiment, the hybrid virus is modified by cloning the rep cDNA directly into the adenovirus genome portion of the hybrid vector. These and additional aspects of this hybrid vector are provided by the above-incorporated by reference application. C. Production of the Recombinant Viral Particle 1. Helper Viruses/Packaging Cell Lines Depending upon the adenovirus gene content of the plasmid vectors employed to carry the VLDLR minigene, a packaging cell line or a helper adenovirus or both may be necessary to provide sufficient adenovirus gene sequences necessary to produce an infective recombinant viral particle containing the VLDLR minigene. Useful helper viruses contain selected adenovirus gene sequences not present in the adenovirus vector construct or expressed by the cell line in which the vector is transfected. A preferred helper virus is desirably replication defective and contains a variety of adenovirus genes in addition to the modified sequences described above. In this setting, the helper virus is desirably used in combination with a packaging cell line that stably expresses adenovirus genes. Helper viruses may also be formed into poly-cation conjugates as described in Wu et al, J. Biol. Chem.. 264:16985-16987 (1989); K. J. Fisher and J. M. Wilson, Biochem. J.. 299:49 (April 1, 1994), and in U. S. Patent Application Serial No. 08/331,381, incorporated by reference herein. Helper virus may optionally contain a second reporter minigene. A number of such reporter genes are known to the art. The presence of a reporter gene on the helper virus which is different from the transgene on the adenovirus vector allows both the Ad vector and the helper virus to be independently monitored. This second reporter is used to enable separation between the resulting recombinant virus and the helper virus upon purification. The construction of desirable helper cells is within the skill of the art. As one example, if the cell line employed to produce the viral vector is not a packaging cell line, and the vector contains only the minimum adenovirus sequences identified above, the helper virus may be a wild type Ad vector supplying the necessary adenovirus early genes El, E2a, E4 and all remaining late, intermediate, structural and non-structural genes of the adenovirus genome. However, if, in this situation, the packaging cell line is 293, which supplies the El proteins, the helper cell line need not contain the El gene. In another embodiment, if the adenovirus vector construct is replication defective (no El gene and optionally no E3 gene) and the 293 cell line is employed, no helper virus is necessary for production of the hybrid virus. E3 may be eliminated from the helper virus because this gene product is not necessary for the formation of a functioning virus particle. Preferably, to facilitate purification and reduce contamination of the viral vector particle with the helper virus, it is useful to modify the helper virus' native adenoviral gene sequences which direct efficient packaging, so as to substantially disable or "cripple" the packaging function of the helper virus or its ability to replicate. A desirable "crippled" adenovirus is modified in its 5' ITR packaging/enhancer domain, which normally contains at least seven distinct yet functionally redundant sequences necessary for efficient packaging of replicated linear adenovirus genomes ("PAC" sequences) . Within a stretch of nucleotide sequence from bp 194-358 of the Ad5 genome, five of these PAC sequences are localized: PAC I or its complement at bp 241-248 [SEQ ID NO: 4], PAC II or its complement at bp 262-269 [SEQ ID NO: 5], PAC III or its complement at bp 304-311 [SEQ ID NO: 6], PAC IV or its complement at bp 314-321 [SEQ ID NO: 7], and PAC V or its complement at bp 339-346 [SEQ ID NO: 8]. Mutations or deletions may be made to one or more of these PAC sequences in an adenovirus helper virus to generate desirable crippled helper viruses. Modifications of this domain may include 5' adenovirus sequences which contain less than all five of the native adenovirus PAC sequences, including deletions of contiguous or non-contiguous PAC sequences. An alternative modification may be the replacement of one or more of the native PAC sequences with one or more repeats of a consensus sequence containing the most frequently used nucleotides of the five native PAC sequences. Alternatively, this adenovirus region may be modified by deliberately inserted mutations which disrupt one or more of the native PAC sequences. One of skill in the art may further manipulate the PAC sequences to similarly achieve the effect of reducing the helper virus packaging efficiency to a desired level. It should be noted that one of skill in the art may design other helper viruses or develop other packaging cell lines to complement the adenovirus deletions in the vector construct and enable production of the recombinant virus particle, given this information. Therefore, the use or description of any particular helper virus or packaging cell line is not limiting. In the presence of other packaging cell lines which are capable of supplying adenoviral proteins in addition to the El, the helper virus may accordingly be deleted of the genes encoding these adenoviral proteins. Such additionally deleted helper viruses also desirably contain crippling modifications as described above. Poly-cation helper virus conjugates, which may be associated with a plasmid containing other adenoviral genes, which are not present in the helper virus may also be useful. The helper viruses described above may be further modified by resort to adenovirus- polylysine conjugate technology. See, e.g., Wu et al, cited above; and K. J. Fisher and J. M. Wilson, cited above. Using this technology, a helper virus containing preferably the late adenoviral genes is modified by the addition of a poly-cation sequence distributed around the capsid of the helper virus. Preferably, the poly-cation is poly-lysine, which attaches around the negatively-charged vector to form an external positive charge. A plasmid is then designed to express those adenoviral genes not present in the helper virus, e.g., the El, E2 and/or E4 genes. The plasmid associates to the helper virus-conjugate through the charges on the poly-lysine sequence. This conjugate permits additional adenovirus genes to be removed from the helper virus and be present on a plasmid which does not become incorporated into the virus during production of the recombinant viral vector. Thus, the impact of contamination is considerably lessened. 2. Assembly of Viral Particle and Infection of a Cell Line Assembly of the selected DNA sequences of the adenovirus, the AAV and the reporter genes or therapeutic genes and other vector elements into the hybrid vector and the use of the hybrid vector to produce a hybrid viral particle utilize conventional techniques. Such techniques include conventional cloning techniques of cDNA such as those described in texts [Sambrook et al, cited above], use of overlapping oligonucleotide sequences of the adenovirus and AAV genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence. Standard transfection and co-transfection techniques are employed, e.g. , CaP0 4 transfection techniques using the complementation 293 cell line. Other conventional methods employed include homologous recombination of the viral genomes, plaquing of viruses in agar overlay, methods of measuring signal generation, and the like. For example, following the construction and assembly of the desired minigene- containing plasmid vector, the vector is infected in vitro in the presence of an optional helper virus and/or a packaging cell line. Homologous recombination occurs between the helper and the vector, which permits the adenovirus-transgene sequences in the vector to be replicated and packaged into virion capsids, resulting in the recombinant vector viral particles. The current method for producing such virus particles is transfection-based. Briefly, helper virus is used to infect cells, such as the packaging cell line human HEK 293, which are then subsequently transfected with an adenovirus plasmid vector containing a VLDLR transgene by conventional methods. About 30 or more hours post- transfection, the cells are harvested, an extract prepared and the recombinant virus vector containing the VLDLR transgene is purified by buoyant density ultracentrifugation in a CsCl gradient. The yield of transducing viral particles is largely dependent on the number of cells that are transfected with the plasmid, making it desirable to use a transfection protocol with high efficiency. One such method involves use of a poly-L- lysinylated helper adenovirus as described above. A plasmid containing the VLDLR minigene is then complexed directly to the positively charged helper virus capsid, resulting in the formation of a single transfection particle containing the plasmid vector and the helper functions of the helper virus. II . Use of the Recombinant Virus Vectors in Gene Therapy The resulting recombinant adenoviral vector containing the VLDLR minigene produced by cooperation of the adenovirus vector and helper virus or adenoviral vector and packaging cell line, as described above, thus provides an efficient gene transfer vehicle which can deliver the VLDLR gene to a patient in vivo or ex vivo and provide for integration of the gene into a liver cell. The above-described recombinant vectors are administered to humans in a conventional manner for gene therapy and serve as an alternative or supplemental gene therapy for LDL receptor deficiencies or other lipoprotein metabolic disorders. A viral vector bearing the VLDLR gene may be administered to a patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. A suitable vehicle includes sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose. The viral vectors are administered in sufficient amounts to transfect the liver cells and provide sufficient levels of transfer and expression of the VLDLR gene to provide a therapeutic benefit without undue adverse or with medically acceptable physiological effects which can be determined by those skilled in the medical arts. Conventional and pharmaceutically acceptable routes of administration include direct delivery to the liver, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral and other parental routes of administration. Routes of administration may be combined, if desired. Dosages of the viral vector will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients. For example, a therapeutically effective human dosage of the viral vector is generally in the range of from about 20 to about 100 ml of saline solution containing concentrations of from about 1 x 10 9 to 1 x 10 u pfu/ml virus vector. A preferred human dosage is estimated to be about 50 ml saline solution at 2 x 10 10 pfu/ml. The dosage will be adjusted to balance the therapeutic benefit against any adverse side effects. The levels of expression of the VLDLR gene can be monitored to determine the frequency of dosage administration. An optional method step involves the co- administration to the patient, either concurrently with, or before or after administration of the viral vector, of a suitable amount of an immune modulator, which is preferably short-acting. The selected immune modulator is defined herein as an agent capable of inhibiting the formation of neutralizing antibodies directed against products of the recombinant vector of this invention and/or capable of inhibiting cytolytic T lymphocyte (CTL) elimination of the vector containing cells. The immune modulator may interfere with the interactions between the T helper subsets (T HI or T m ) and B cells to inhibit neutralizing antibody formation. Alternatively, the immune modulator may be selected to inhibit the interaction between T H1 cells and CTLs to reduce the occurrence of CTL elimination of the vector. More specifically, the immune modulator desirably interferes with, or blocks, the function of the CD4 T cells. Immune modulators for use in inhibiting neutralizing antibody formation may be selected based on the determination of the immunoglobulin subtype of any neutralizing antibody produced in response to the VLDLR- containing adenovirus vector. For example, if the neutralizing antibody is a T m mediated antibody, such as IgA, the immune modulator desirably suppresses or prevents the interaction of T m with B cells. Alternatively, if the induced neutralizing antibody is a T HI mediated antibody, such as IgG^, the immune modulator desirably suppresses or prevents the interaction of T H1 with B cells. The neutralizing antibody which develops in response to administration of a viral vector of this invention can be based on what vehicle is being used to deliver the vector and/or the location of delivery. For instance, administration of adenoviral vectors via the lungs generally induces production of IgA neutralizing antibody. Administration of adenoviral vectors via the blood generally induces IgG, neutralizing antibody. The determination of the neutralizing antibody is readily determined in trials of the selected viral vector in animal models. Where the reduction of CTL elimination of the viral vectors is desired, the immune modulator is selected for its ability to suppress or block CD4 + T HI cells to permit prolonged residence of the viral vector in vitro. Selection of the immune modulator thus may be based upon the mechanism sought to be interrupted or blocked. The immune modulators may be soluble proteins or naturally occurring proteins, including cytokines, monoclonal antibodies. The immune modulators may be conventional pharmaceuticals. The immune modulators identified herein may be used alone or in combination with one another. For example, cyclophosphamide and the more specific immune modulator anti-CD4 monoclonal antibody may be co-administered. In such a case, cyclophosphamide serves as an agent to block T H1 activation and stabilized transgene expression beyond the period of transient immune blockade. A suitable amount or dosage of the immune modulator will depend primarily on the amount of the recombinant vector bearing the VLDLR gene which is initially administered to the patient and the type of immune modulator selected. Other secondary factors such as the condition being treated, the age, weight, general health, and immune status of the patient, may also be considered by a physician in determining the dosage of immune modulator to be delivered to the patient. Generally, for example, a therapeutically effective human dosage of a cytokine immune modulator, e.g., IL-12 or 7-IFN, is generally in the range of from about 0.5 μg to about 5 mg per about 1 x 10 7 pfu/ml virus vector. Various dosages may be determined by one of skill in the art to balance the therapeutic benefit against any side effects. A. Monoclonal Antibodies and Soluble Proteins Preferably, the method of inhibiting an adverse immune response to the gene therapy vector involves non-specific inactivation of CD4 + cells. Preferably, such blocking antibodies are "humanized" to prevent the recipient from mounting an immune response to the blocking antibody. A "humanized antibody" refers to an antibody having its complementarily determining regions (CDRs) and/or other portions of its light and/or heavy variable domain framework regions derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulins. Such antibodies can also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa. Such "humanization" may be accomplished by methods known to the art. See, for example, G.E. Mark and E. A. Padlan, "Chap. 4 . Humanization of Monoclonal Antibodies" , The Handbook of Experimental Pharmacology, vol. 113, Springer-Verlag, New York (1994), pp. 105-133, which is incorporated by reference herein. Other suitable antibodies include those that specifically inhibit or deplete CD4 + cells, such as an antibody directed against cell surface CD4. Depletion of CD4 + cells has been shown by the inventors to inhibit the CTL elimination of the viral vector. Such modulatory agents include but are not limited to anti-T cell antibodies, such as anti-OKT3+ [see, e.g., US Patent No. 4,658,019; European Patent Application No. 501,233, published September 2, 1992]. See Example 2 below, which employs the commercially available antibody GK1.5 (ATCC Accession No. TIB207) to deplete CD4 + cells. Alternatively, any agent that interferes with or blocks the interactions necessary for the activation of B cells by T H cells, and thus the production of neutralizing antibodies, is useful as an immune modulator according to these methods. For example, B cell activation by T cells requires certain interactions to occur [F. H. Durie et al, Immunol. Today. 15(9) :406-410 (1994)], such as the binding of CD40 ligand on the T helper cell to the CD40 antigen on the B cell, and the binding of the CD28 and/or CTLA4 ligands on the T cell to the B7 antigen on the B cell. Without both interactions, the B cell cannot be activated to induce production of the neutralizing antibody. The CD40 ligand (CD40L)-CD40 interaction is a desirable point to block the immune response to gene therapy vectors because of its broad activity in both T helper cell activation and function as well as the absence of redundancy in its signaling pathway. A currently preferred method of the present invention thus involves transiently blocking the interaction of CD40L with CD40 at the time of adenoviral vector administration. This can be accomplished by treating with an agent which blocks the CD40 ligand on the T H cell and interferes with the normal binding of CD40 ligand on the T helper cell with the CD40 antigen on the B cell. Blocking CD40L-CD40 interaction prevents the activation of the T helper cells that contributes to problems with transgene stability and readministration. Thus, an antibody to CD40 ligand (anti- CD40L) [available from Bristol-Myers Squibb Co; see, e.g., European patent application 555,880, published August 18, 1993] or a soluble CD40 molecule can be a selected immune modulator in this method. Alternatively, an agent which blocks the CD28 and/or CTLA4 ligands present on T helper cells interferes with the normal binding of those ligands with the antigen B7 on the B cell. Thus, a soluble form of B7 or an antibody to CD28 or CTLA4, e.g., CTLA4-Ig [available from Bristol-Myers Squibb Co; see, e.g., European patent application 606,217, published July 20, 1994] can be the selected immune modulator in the method of this invention. This method has greater advantages than the below-described cytokine administration to prevent T m activation, because it addresses both cellular and humoral immune responses to foreign antigens. B . Cytokines Still other immune modulators which inhibit the T H cell function may be employed in this invention. Thus, in one embodiment, an immune modulator which selectively inhibits the function of the T H , subset of CD4 + T helper cells may be administered at the time of primary administration of the viral vector. One such immune modulator is interleukin-4 (IL-4) . IL-4 enhances antigen specific activity of T m cells at the expense of the T H , cell function [see, e.g., Yokota et al, Proc. Natl. Acad. Sci.. USA. £2:5894-5898 (1986); United States Patent No. 5,017,691]. It is envisioned that other immune modulators that can inhibit T H1 cell function will also be useful in the methods of this invention. In another embodiment, the immune modulator can be a cytokine that prevents the activation of the Tr ø subset of T helper cells. The success of this method depends on the relative contribution that T^ dependent Ig isotypes play in virus neutralization, the profile of which may be affected by strain, the species of animal as well as the mode of virus delivery and target organ. A desirable immune modulator which selectively inhibits the CD4 + T cell subset T ffi function at the time of primary administration of the viral vector includes interleukin-12 (IL-12) . IL-12 enhances antigen specific activity of T H1 cells at the expense of T m cell function [see, e.g., European Patent Application No. 441,900; P. Scott, Science. 260:496-497 (1993); R. Manetti et al, J. EXP. Med.. 177:1199 (1993); A. D'Andrea et al, J. EXP. Med.. 176:1387 (1992)]. IL-12 for use in this method is preferably in protein form. Human IL-12 may be recombinantly produced using known techniques or may be obtained commercially. Alternatively, it may be engineered into a viral vector (which optionally may be the same as that used to express the transgene) and expressed in a target cell in vivo or ex vivo. Trø specific ablation with IL-12 is particularly effective in lung-directed gene therapies where IgA is the primary source of neutralizing antibody. In liver-directed gene therapy, both T H , and T^ cells contribute to the production of virus specific antibodies. However, the total amount of neutralizing antibody can be diminished with IL-12. Another selected immune modulator which performs a similar function is gamma interferon (IFN-7) [S. C. Morris et al, J. Immunol.. 151:1047-1056 (1994); F. P. Heinzel et al, J. EXP. Med.. 177:1505 (1993)]. IFN-7 is believed to mediate many of the biological effects of IL-12 via secretion of activated macrophages and T helper cells. IFN-γ also partially inhibits IL-4 stimulated activation of T^. IFN-7 may also be obtained from a variety of commercial sources. Alternatively, it may be engineered into a viral vector and expressed in a target cell in vivo or ex vivo using known genetic engineering techniques. Preferably, such cytokine immune modulators are in the form of human recombinant proteins. These proteins may be produced by methods extant in the art. Active peptides, fragments, subunits or analogs of the known immune modulators described herein, such as IL- 12 or gamma interferon, which share the T^ inhibitory function of these proteins, will also be useful in this method when the neutralizing antibodies are T ffi mediated. C. Other Pharmaceuticals Other immune modulators or agents that non-specifically inhibit immune function, i.e., cyclosporin A or cyclophosphamide, may also be used in the methods of the invention. For example, a short course of cyclophosphamide has been demonstrated to successfully interrupt both CD4 and CD8 T helper cell activation to adenovirus capsid protein at the time of virus delivery to the liver. As a result, transgene expression was prolonged and, at higher doses, formation of neutralizing antibody was prevented, allowing successful vector readministration. In the lung, cyclophosphamide prevented formation of neutralizing antibodies at all doses and stabilized transgene expression at high dose. D. Administration of Immune Modulator The optional administration of the selected immune modulator may be repeated during the treatment with the recombinant adenovirus vector carrying the human VLDLR gene, during the period of time that the VLDLR gene is expressed (as monitored by e.g., LDL levels) , or with every booster of the recombinant vector. Thus, the compositions and methods of this invention provide a desirable treatment for defects in LDL metabolism, by providing stable expression of the VLDLR gene in human hepatocytes, and the ability to re- administer the vector as desired without incurring an undesired immune response by the patient. The following examples illustrate the construction and testing of the viral vectors and VLDL receptor gene inserts of the present invention and the use thereof in the treatment of metabolic disorders. An exemplary recombinant adenovirus encoding the human VLDL receptor was constructed as described in Example 1 below. These examples are illustrative only, and do not limit the scope of the present invention. Example 1 - Construction and Purification of HS.OIOCNW&PE The cDNA for the human very low density lipoprotein (VLDL) receptor [M. E. Gafvels et al, cited above; SEQ ID NO: 1] was inserted into the polylinker region of plasmid pRc/CMV (obtained from Invitrogen Corp.). The resulting plasmid, pRc/CMWLDLR, was digested with the restriction enzymes SnaBI and Notl and the 4 kb fragment containing the cytomegalovirus (CMV) immediate-early promoter and VLDL receptor cDNA was isolated. The plasmid pAd.CMVlacZ [Kozarsky II, cited above] was digested with SnaBI and Notl to remove the CMV promoter and lacZ cDNA and the 5.6 kb backbone was isolated. The two fragments were ligated to generate pAd.CMWLDLR (Figs. 2 and 9; SEQ ID NO: 3). pAd.CMWLDLR was linearized with Nhel and co-transfected into 293 cells with sub360 DNA (derived from adenovirus type 5) which had been digested with Xbal and Clal as previously described [K. F. Kozarsky I and II cited above]. The resulting recombinant adenovirus, designated H5.010CMWLDLR, contains the sequence from about nucleotide 12 to about 4390 of pAd.CMWLDLR and Ad.5 map units 9-100 with a small deletion in the E3 gene (see GenBank Accession No. M73260 and discussion of Fig. 3) . This recombinant adenovirus was isolated following two rounds of plaque purification. H5.010CMWLDLR was grown on 293 cells and purified by two rounds of cesium chloride density centrifugation as previously described [K. F. Kozarsky I and II cited above] . Cesium chloride was removed by passing the virus over a BioRad 10DG desalting column equilibrated with phosphate-buffered saline. For rabbit experiments, virus was used freshly purified; for mouse experiments, virus was either used fresh, or after column purification glycerol was added to a final concentration of 10% (v/v) , and virus was stored at -70°C until use. As described in the following examples, this recombinant adenovirus vector was introduced into the livers of WHHL rabbits and into the livers of LDL receptor knockout mice to determine the in vivo function of the VLDL receptor, and to determine its usefulness as an alternative or supplemental gene therapy for LDL receptor deficiency. Example 2 - Other Recombinant Adenoviruses H5.OlOCMVlacZ, encoding the lacZ gene under the control of the CMV enhancer/promoter, and H5.010CBhLDLR, encoding the human low density lipoprotein (LDL) receptor cDNA under the control of the CMV-enhanced chicken β- actin promoter, were prepared as previously described [K. F. Kozarsky I and II, cited above]. Example 3 - Effects of Hepatic Expression of the VLDL Receptor in the WHHL Rabbit H5.010CMWLDLR or H5.OlOCMVlacZ (encoding β- galactosidase) , obtained as described in Examples 1 and 2, was infused intravenously into WHHL rabbits [Camm Research] as follows. Rabbits were infused with 7.5 x 10 12 particles of either recombinant adenovirus through a marginal ear vein on day 0. In addition, two New Zealand White (NZW) rabbits [Hazleton, Inc.] were infused with each virus and sacrificed on day 5 post-infusion to document the extent of gene transfer in the liver. Rabbits were maintained in a 12 hour light/dark cycle on a diet of Purina laboratory chow, delivered each day at approximately 11:00 am. Venous samples were obtained through a marginal ear vein at approximately 10:00 am on the days indicated. A. Plasma Analyses Plasma samples were analyzed for total cholesterol using the Cholesterol HP kit and Precise standards (Boehringer Mannheim) . Briefly, FPLC analysis was performed on 50 μl of plasma from individual mice adjusted to a volume of 250 μl in FPLC column buffer (1 mM EDTA, 154 mM NaCl, pH 8.0). Diluted samples (200 μl) were loaded onto two Superose 6 columns (Pharmacia) in series at a flow rate of 0.4 ml/min, and 1 ml fractions were collected. Cholesterol content was analyzed in a microplate assay on 100 μl samples. 100 μl of a freshly prepared solution containing 50 mM PIPES, pH 6.9, 7.8 g/L HDCBS, 0.51 g/L 4-AAT, 1.27 g/L cholic acid, 0.245% Triton X-100, 7.31 g/L KCl and supplemented with 1.22 U/ml cholesterol oxidase, 7.64 U/ml cholesterol esterase, and 245 U/ml peroxidase was added to samples, incubated overnight at room temperature, and the O.D. at 490 nm was determined. Plasma cholesterol levels were evaluated in each of the WHHL rabbits before and after receiving recombinant adenovirus. Fig. 4A shows that rabbits infused with H5.OlOCMVlacZ had no significant changes in cholesterol levels. However, following infusion with H5.010CMWLDLR, cholesterol levels dropped, with maximum decreases that ranged from 140 to 420 mg/dl (Fig. 4B) . This demonstrated that expression of the VLDL receptor results in decreased cholesterol levels in LDL receptor- deficient rabbits. B . Histoche ical Analysis Portions of liver were paraffin embedded, sectioned, and stained with hematoxylin and eosin. Some portions were fresh-frozen, sectioned, fixed in glutaraldehyde, stained with X-gal and lightly counterstained with hematoxylin. Some fresh-frozen sections were fixed in methanol, and then stained with either a polyclonal anti-0-galactosidase antibody (5 prime-3 prime) , a polyclonal anti-human LDL receptor antibody, or with a polyclonal anti-VLDL receptor antibody, followed by a fluorescein isothiocyanate- conjugated anti-rabbit antibody (Jackson Immunoresearch) as previously described [K. F. Kozarsky I and II cited above] . Oil Red O staining was performed on fresh-frozen sections fixed for 1 minute in 37% formaldehyde, then rinsed and stained in Oil Red O (3 parts 0.5% Oil Red O in isopropyl alcohol/2 parts water) for 10 minutes. Slides were counterstained in hematoxylin and mounted in aqueous solution. Immunofluorescence analysis of the infused rabbits showed that approximately 50% of hepatocytes from the rabbit infused with H5.OlOCMVlacZ expressed /3- galactosidase, liver tissue from the rabbit infused with H5.010CMWLDLR had a slightly higher percentage of hepatocytes expressing the VLDL receptor. In agreement with Northern blot analysis showing little or no VLDL receptor mRNA expression [M. E. Gafvels et al, cited above] , liver from the lacZ-infused rabbit showed no reactivity with the anti-VLDL receptor antibody. Example 4 - Effects of Short-Term Hepatic Expression of the VLDL Receptor in LDL Receptor Knockout Mice C57BL/6 mice and LDL receptor knockout mice (Jackson Labs) were infused intravenously with 0.5 or 1.0 x 10 10 particles of recombinant adenovirus through the tail vein and cholesterol levels were monitored before and after infusion. Specifically, three mice each were infused with either H5.OlOCMVlacZ, H5.010CMWLDLR, or H5.OlOCBhLDLR (encoding the human LDL receptor cDNA) . This last virus was included as a control to confirm published results [Kozarsky I and II cited above] . Plasma samples were obtained by retro-orbital bleeds using heparinized capillary tubes. The LDL receptor knockout mice were maintained upon a high cholesterol diet composed of Purina mouse chow supplemented with 1.25% cholesterol, 7.5% cocoa butter, 7.5% casein, and 0.5% cholate (1.25% cholesterol diet) for at least 3 weeks immediately following weaning before experiments were initiated. Mice were sacrificed on day 5 post-infusion. Liver tissues were analyzed by immunofluorescence for transgene expression by the techniques described in Example 3, and plasma cholesterol levels were measured as similarly described. For lipoprotein fractionations, plasma from triplicate LDL receptor knockout mice were pooled, subjected to density ultracentrifugation, fractions were collected, and the cholesterol content was determined by conventional means. Immunofluorescence analysis revealed moderate levels of S-galactosidase expression in H5.010CMVlacZ- infused mice, and higher levels of either human LDL receptor and VLDL receptor expression in H5.010CBhLDLR- and in H5.010CMWLDLR-infused mice, respectively. Cholesterol levels decreased slightly in the control, H5.010CMVlacZ-infused mice (Fig. 5), probably due to non-transgene-related effects of infusion of recombinant adenovirus, which can result in hepatotoxicity in mice [Y. Yang et al, Proc. Natl. Acad. Sci.. USA. 21:4407-4411 (May 1994)]. However, in contrast to the decrease observed in the control mice, cholesterol levels dropped significantly to 50% of pre- infusion values in the H5.010CBhLDLR-infused mice on day 5 post-infusion. Cholesterol levels in the H5.010CMWLDLR-infused mice also decreased, to approximately 60% of pre-infusion levels. Further analysis of plasma lipoproteins showed that in the H5.010CBhLDLR-treated mice, LDL levels plummeted, with additional decreases in IDL and VLDL fractions (Fig. 6) . The H5.010CMWLDLR-infused mice showed a larger decrease in the VLDL fraction with less of a decrease in LDL . Taken together, these data indicate that hepatic expression of VLDL receptor results in increased clearance of VLDL from the plasma, resulting in decreases in the amounts of lipoproteins for which VLDL is the precursor (i.e., IDL and LDL) , and an overall drop in total plasma cholesterol. Example 5 - Effects of Long-Term Hepatic Expression of the VLDL Receptor in LDL Receptor Knockout Mice In order to achieve cholesterol levels closer to those observed in both FH patients and WHHL rabbits, LDL receptor knockout mice (Jackson Labs) were maintained on a high cholesterol diet composed of Purina mouse chow supplemented with 0.2% cholesterol, 10% coconut oil, and 0.05% cholate (0.2% cholesterol diet). Cholesterol levels in these mice ranged from 930 to 1550 mg/dl, whereas the mice on the 1.25% cholesterol (Example 4) diet had levels of 1900 to 3100 mg/dl. Virus was thawed immediately before use and diluted with PBS to a concentration of lxio 12 particles/ml. Three mice were each infused intravenously with 0.1 ml of virus containing 1 x 10" particles of an El-deleted recombinant adenovirus encoding either β- galactosidase (H5.OlOCMVlacZ) or human LDL receptor (H5.010CBhLDLR) , and serum lipids were followed over time. On the days indicated, mice were anesthetized with methoxyflurane and blood was collected into heparinized capillary tubes by puncture of the retro-orbital venous plexus. Im unofluorescence staining showed that most of the hepatocytes expressed the transgene product, either 3-galactosidase, human LDL receptor, or VLDL receptor. Hematoxylin and eosin staining of sections of liver revealed essentially normal morphology in the H5.010CMVlacZ-infused mouse. However, for both the H5.010CBhLDLR- and H5.OlOCMWLDLR-infused mice, hepatocytes appeared to have internal vacuoles. When tissue was analyzed with Oil Red O staining, a stain for neutral lipids, liver from the receptor-infused animals clearly showed accumulation of large droplets of lipid when compared with the H5.010CMVlacZ-infused control. This suggested that short-term, high level expression of the LDL receptor or VLDL receptor in these LDL receptor- deficient mice resulted in intracellular accumulation of lipids. To confirm the biological activities of the transgene products, plasma cholesterol levels were followed before and after recombinant adenovirus administration. Fig. 7A shows that serum cholesterol levels in H5.OlOCMVlacZ-infused mice demonstrated a characteristic but not significant fluctuation over time, reflected in minor changes of all lipoprotein fractions (HDL, IDL/VLDL, and LDL) . In contrast, mice infused with H5.OlOCBhLDLR have a large but transient decrease in cholesterol (see, Fig. 7B) . Particularly, these mice demonstrated large plasma cholesterol decreases which lasted for approximately 2 weeks. Cholesterol levels decreased 3-fold (from 966 to 353 mg/dl) and 7-fold (from 1554 to 219 mg/dl) and returned to baseline by 3 weeks post-infusion. The decrease in serum cholesterol is reflected in coordinate diminution in serum LDL. This nonspecific effect of the adenovirus infection when immune modulators are not coordinately administered has been described previously and is likely due to changes in hepatic function that occur as a result of the associated inflammation. Mice infused with H5.010CMWLDLR showed large decreases in plasma cholesterol which were similar in magnitude to those seen in the H5.010CBhLDLR-infused mice (Fig. 7C) , with maximum decreases of more than 4- fold (from 1186 to 288 mg/dl and from 1453 to 299 mg/dl) . Surprisingly, plasma cholesterol levels did not return to baseline by 3 weeks post-infusion. The change in plasma cholesterol levels in the H5.010CMWLDLR-infused mice (Fig. 7A) were statistically significant (p<0.05) through 9 weeks following infusion (the current duration of the experiment) . Sera from individual mice was analyzed by FPLC to determine the effects of VLDL receptor expression on lipoprotein fractions. On day 3 post-infusion, VLDL and LDL fractions were undetectable; over time, the LDL fraction slowly recovered, although even at 10 weeks post-infusion, the LDL peak height was slightly lower than the HDL peak height. VLDL remained undetectable although minor differences may escape detection because of limitations in the sensitivity of the cholesterol assay. The LDL peaks mirrored the total plasma cholesterol levels, and confirmed that the prolonged lowering of plasma cholesterol was accompanied by sustained decreases in VLDL and LDL levels. These data suggest that expression of the VLDL receptor in the liver is an effective therapy for hypercholesterolemia. At the same time of infusion of the LDL receptor knockout mice, normal C57B1/6 mice were infused with each of the recombinant adenoviruses. These mice were sacrificed at various times post-infusion, and liver tissues were harvested for direct analysis of transgene expression using X-gal histochemistry to detect β- galactosidase expression and immunofluorescence performed to measure LDL receptor expression. Tissues harvested three days after infusion of virus demonstrated either expression of β-galactosidase or the human LDL receptor in at least 80% of hepatocytes. In each experiment, the vector specific signal was substantially higher than that seen in animals before gene transfer or following infusion with identical quantities of an adenovirus expressing an irrelevant gene. For both lacZ and LDL receptor, transgene expression diminished to undetectable levels by day 21 and was associated with the development of a self limited mononuclear infiltrate in liver that peaked at day 10. The infiltrate consisted of portal as well as lobular inflammation, accompanied by the presence of apoptic bodies. The extent of pathology was indistinguishable between the lacZ and LDL receptor infused mice. The time course of LDL receptor expression is consistent with the initial large decline in plasma cholesterol and subsequent return to baseline. In contrast, two mice infused with H5.010CMWLDLR expressed the VLDL receptor at high levels. The percent of hepatocytes may have decreased slightly as compared to the day 5 mice. These data suggest that the sustained decrease in plasma cholesterol levels in the H5.010CMWLDLR-infused mice was due to sustained expression of the VLDL receptor. Example 6 - Turnover Studies To further characterize the effects of hepatic VLDL receptor expression on lipoprotein metabolism, turnover studies were performed as follows. LDL receptor knockout mice were infused with recombinant adenovirus after 3 weeks on the high cholesterol diet as described in Example 4. Three mice each were injected with the lacZ and VLDL receptor adenoviruses; one mouse was injected with the LDL receptor adenovirus. On day 5 post-infusion, mice were injected via the tail vein with approximately 8 x 10 6 cpm of I25 I-labeled human LDL, and 1.6 x 10 s cpm of 13, I-labeled human VLDL in a total volume of 0.2 ml. A blood sample was obtained 1 minute following injection of radiolabel, and designated the "time zero" sample. Blood was collected into heparinized capillary tubes at the indicated times, and radioactivity remaining was determined using a gamma counter. Infusion of LDL receptor adenovirus led to accelerated clearance of LDL as compared to infusion of lacZ adenovirus, consistent with a previous study in LDL receptor knockout mice [S. Ishibashi et al, J. Clin. Invest.. 22:883-893(1993)]. Similarly, VLDL clearance was accelerated in LDL receptor treated animals as compared to lacZ infused mice. LDL turnover in VLDL receptor-infused mice was indistinguishable from lacZ infused mice, consistent with in vitro data which indicates that LDL is not a ligand for the VLDL receptor [T. Yamamoto et al, Trends in Cardiovascular Medicine. 2:144-148 (1993); F. Batley et al, J. Biol. Chem.. 2^2:23268-23273 (1994)]. VLDL clearance in VLDL receptor infused mice was slightly faster than n lacZ infused mice, but significantly slower than in LDL receptor infused mice. As discussed above, VLDL turnover in mice infused with the VLDL receptor adenovirus was significantly faster than in lacZ infused mice although the magnitude of this effect was far less than that seen in animals treated with LDL receptor virus. This suggests that VLDL receptor-mediated clearance of circulating VLDL may not be the only pathway leading to diminished serum VLDL. One potential mechanism is secretion-recapture, which occurs with hepatic uptake of chylomicron remnants [T. Willnow & J. Herz, J. Mol. Med.. 22:213-220 (1995); H. Shimano et al, J. Clin. Invest.. 22:2215-2223 (1994)], and would result in decreased secretion of VLDL and reduced levels of plasma VLDL. A second mechanism may involve the interaction of the VLDL receptor with receptor-associated protein (RAP) [Battey, cited above; H. Mokuno et al, J. Biol. Chem.. 269:13238- 13243 (1994)] which interacts with a variety of receptors inside the cell, apparently to prevent ligand binding before the receptor reaches the cell surface [G. Bu et al, EMBO J. 14:2269-2280 (1995)]. It is possible that the high levels of VLDL receptor expressed in the livers of adenovirus-infused mice overwhelms the available RAP, so that VLDL receptor is binding to newly synthesized ligand (apoE, either free or in association with lipid) within the cell, and preventing its secretion into the plasma. The effects of hepatic VLDL receptor expression on total plasma cholesterol as well as on lipoprotein cholesterol levels demonstrate that the VLDL receptor can play a major role in lipoprotein metabolism in vivo. Example 7 - stability of Expression of VLDL Receptor This experiment illustrates relative transgene persistence in mice. LDL receptor knockout mice were injected intravenously on day 0 with lxlO 1 ' particles of H5.OlOCMVlacZ, H5.OlOCBhLDLR, or H5.010CMWLDLR. Mice were sacrificed on the indicated days after injection (3, 10 or 21) , and fresh-frozen sections of liver were stained with X-gal to detect expression of the lacZ gene, and with anti-LDL receptor antibody or anti-VLDL receptor antibody, followed by a fluorescein-conjugated secondary antibody to detect LDL receptor and VLDL receptor, respectively. Analysis of liver harvested 3 days after infusion of virus revealed VLDL receptor protein in >80% of hepatocytes; the bright fluorescent signal, which localized to the perimeter of the cell, was absent before gene transfer and in tissues of animals infected with lacZ or LDL receptor containing adenoviruses. Expression of VLDL receptor protein was remarkably stable with recombinant protein detected in approximately 5 to 10% of hepatocytes from tissue harvested 105 days after infusion of virus. This is in striking contrast to the results obtained with lacZ and LDL receptor adenovirus, where expression of the transgene extinguished to undetectable levels within three weeks of gene transfer. VLDL receptor expression remained detectable through the duration of the experiment (22 weeks) . Genomic DNA was isolated from mouse liver, digested with EcoRI, and subjected to Southern blotting [K. Kozarsky et al. J. Biol. Chem.. 2_69:13695-13702 (1994)] to monitor the presence over time of adenoviral DNA sequences. Adenovirus sequences were detected using the Genius kit from Boehringer Mannheim, followed by chemiluminescent detection. In C57BL/6 mice infused with the lacZ adenovirus, viral DNA diminished rapidly with time, plateauing at barely detectable levels ('0.05 copies/cell) through day 70 post-infusion. Mice infused with VLDL receptor had slightly higher initial levels of DNA, but a similar time course of loss of adenovirus sequences. Additional DNA hybridization studies showed that the majority of adenovirus DNA initially delivered to the liver is not integrated into the mouse genome (data not shown) , however, this assay cannot rule out some level of integration. Histopathologic analysis of liver tissue from mice infused with the VLDL receptor virus revealed inflammation and apoptotic cells at early time points. The timing and extent of the pathologic findings were indistinguishable from liver tissues of mice infused with lacZ and LDL receptor viruses. At 15 and 22 weeks post- infusion, however, liver tissue from VLDL receptor- infused mice displayed discernible accumulations of neutral lipids, as demonstrated by hematoxylin and eosin as well as oil red O staining. Similar changes were observed infrequently in LDL receptor knockout mice infused with PBS, LDL receptor and/or lacZ adenoviruses. No lipid accumulations were observed in livers of normal C57BL/6 mice infused with the VLDL receptor virus, despite long-term transgene expression indistinguishable from that observed in LDL receptor knockout mice. This indicates that VLDL receptor expression alone is not sufficient for the changes in lipid accumulation observed in LDL receptor knockout mice; instead, there is some lipid accumulation in the LDL receptor knockout mice which have been maintained on a high cholesterol diet for > 18 weeks, that is accelerated by prolonged VLDL receptor expression. Plasma samples from mice infused with VLDL receptor adenovirus were analyzed for the presence of antibodies directed against the VLDL receptor protein. Only one mouse out of twelve generated antibodies to the VLDL receptor despite the presence of high level antibodies to adenovirus capsid proteins in each animal that received virus. Animals infused with the VLDL receptor adenovirus mounted a CTL response to adenoviral proteins indistinguishable from that obtained from animals infused with either lacZ or LDL receptor adenoviruses. These mice, however, did not mount a CTL response to the VLDL receptor protein. Thus, the development of a CTL response to the transgene following infusion of recombinant adenovirus is dependent on the antigenicity of the specific transgene in the treated animal. Example 8 - Humoral and Cellular Immune Response to Adenovirus and Transgenes A. Humoral Immune Response Two LDL receptor knockout mice (K020 and K027) or two normal C57BL/6 mice were injected via the tail vein with 1x10" particles of H5.0l0CBhLDLR at day 0 and serum samples were collected both before injection (pre), and on days 10, 24, 39, 52 and 70 following injection for the knockout mice and on day 21 for the C57BL/6 mice. Western blots were performed as previously described [K. Kozarsky et al, J. Biol. Chem.. 269:13695- 13702 (1994); K. Kozarsky et al., Som. Cell and Molec. Genet.. 12:449-458 (1993)]. To detect anti-adenovirus antibodies, purified adenovirus was used as the antigen. The positive control (+) was rabbit antiserum isolated following intravenous infusion of purified H5.010CBhLDLR. The negative control (-) was pre-immune rabbit serum. Western blots with β-galactosidase were performed using purified protein (Sigma) , with a monoclonal antibody specific for β-galactosidase (Sigma) as a positive control. Antibodies directed against the human LDL receptor were detected using lysates prepared from 24-23 cells, a 3T3 cell line which was transduced with retrovirus encoding the human LDL receptor. For detection of anti-VLDL receptor antibodies, a lysate was prepared from HeLa cells two days following infection With H5.010 CMWLDLR. All mice infused with lxio" particles of recombinant adenovirus developed antibodies to adenovirus capsid proteins, with major bands corresponding to hexon, penton and fiber. All mice infused with H5.010CBhLDLR developed antibodies to the human LDL receptor protein with LDL receptor knockout mice consistently developing higher titer antibodies that C57BL/6 mice. Antibodies from LDL receptor knockout mice cross-reacted with mouse LDL receptor protein, whereas antibodies from C57BL/6 mice (which express normal mouse LDL receptor) did not. This suggests that the VLDL receptor, although the human and not the mouse sequence was used, was not immunogenic in these mice. The amino acid sequences of the human and mouse LDL receptors are approximately 78% identical, while the human and mouse VLDL receptors are >94% identical. This increased sequence similarity is likely to account for the absence of antibody development to the human VLDL receptor despite high level expression in the mouse liver as a result of infusion of H5.010CMWLDLR. These data demonstrate that animals can generate a humoral immune response specific for the transgene product as well as to the viral proteins encoded on the injected adenovirus. It also provides indirect evidence of antigen specific activation of T helper cells, which is normally required for development of mature, antibody-secreting B cells. B . Cellular Immune Responses This study analyzed animals following infusion with the LDL receptor adenovirus for activation of CTLs to both viral antigens and the transgene product, human LDL receptor. CTL assays were performed as described in Y. Yang et al, Immunity. 1:433-442 (1994). Target cells expressing recombinant vaccinia proteins were generated by infecting with recombinant vaccinia were generated as follows. The VLDV receptor CDNA (in the pRC/CMV plasmid) was subcloned into the Hindlll site of Bluescript KS+. The CFTR cDNA [J.R. Riordan et al, Science. 245:1066-1073 (1989) was cloned into the Pstl site of Bluescript KS+ (Stratagene) . The LDL receptor cDNA in the pUC19 vector [T. Yamamoto et al, Cell. 22:27-38 (1984)] was excised with the restriction enzymes Hindlll and Sac 1 and ligated into the Hindlll and Sacl sites of Bluescript KS+. Each of the cDNAs was then excised using the enzymes SacII and Kpnl and cloned into the SacII and Kpnl sites of a modified form of the vaccinia expression vector pSCll [S. Chakrabarti et al, Molec. Cell. Biol.. 5:3403-3409 (1985)]. The control recombinant vaccinia, VRG, expresses a rabies virus glycoprotein and was prepared as described in T. Wiktor et al, Proc. Natl. Acad. Sci. USA. 21: 7194-7198 (1984). CTLs to specific targets were detected in a standard 51 chromium ( 5l Cr) release assay in which MHC compatible target cells were infected with either recombinant adenovirus or vaccinia viruses that express single relevant gene products. Figure 10 presents both an example of a 5l Cr release assay in which % specific lysis is measured as a function of increasing the effector to target ratio (Fig. 10B) , as well as a summary of the cumulative data (Fig. 10A) . Splenocytes from C57BL/6 mice infused with recombinant adenovirus containing either human LDL receptor or human CFTR were evaluated for their ability to lyse targets infected with either recombinant adenovirus, to measure activity to viral proteins, or with vaccinia virus containing LDL receptor, to measure activity to LDL receptor protein. Cytolytic activity was demonstrated with lymphocytes from animals infected with the LDL receptor virus to target cells infected with the same virus. No cytolysis was detected to mock infected targets supporting the specificity of the assay. These same effector cells demonstrated significant cytolytic activity to targets infected with LDL receptor vaccinia virus that was not present when infected with a control vaccinia. These experiments provide strong evidence for the presence of activated CTL to human LDL receptor in C57BL/6 mice following gene therapy. Example 9 - Enhancement of Adenovirus Mediated Gene Transfer upon Second Administration by IL-12 and IFN-τ in Mouse Lung. The recombinant adenoviruses H5.OlOCMVlacZ and H5.010CBALP (alkaline phosphatase gene expressed from the CMV enhanced β-actin promoter in the sub360 backbone) were used in this example. Each similar virus expresses a different reporter gene whose expression can be discriminated from that of the first reporter gene. Female C57B1/6 mice (6-8 week old) were infected with suspensions of H5.010CBALP (1 x 10 9 pfu in 50 μl of PBS) via the trachea at day 0 and similarly with H5.OlOCMVlacZ at day 28. One group of such mice was used as a control. Another group of mice were acutely depleted of CD4 + cells by i.p. injection of antibody to CD4 + cells (GK1.5; ATCC No. TIB207, 1:10 dilution of ascites) at the time of the initial gene therapy (days - 3, 0, and +3). A third group of mice were injected with IL-12 (1 μg intratracheal or 2 μg, i.p. injections) at the time of the first administration of virus (days 0 and +1) . A fourth group of mice were injected with gamma interferon (1 μg intratracheal or 2 μg, i.p. injections) at the time of the first administration of virus (days 0 and +1) . When mice were subsequently euthanized and necropsied at days 3, 28, or 31, lung tissues were prepared for cryosections, while bronchial alveolar lavage (BAL) and mediastinal lymph nodes (MLN) were harvested for immunological assays. A. Cryosections The lung tissues were evaluated for alkaline phosphatase expression by histochemical staining following the procedures of Y. Yang et al, cited above. Instillation of alkaline phosphatase virus (10 9 pfu) into the airway of all groups of the C57B1/6 mice resulted in high level transgene expression in the majority of conducting airways that diminishes to undetectable levels by day 28. Loss of transgene expression was shown to be due to CTL mediated elimination of the genetically modified hepatocytes [Y. Yang et al, cited above]. In the control mice, no recombinant gene expression was detected three days after the second administration of virus, i.e., day 31. Administration of virus to the CD4 + depleted animals was associated with high level recombinant transgene expression that was stable for a month. Expression of the second virus was detectable on day 31. Initial high level gene transfer diminished after about one month in the IL-12 treated mice; however, in contrast to the control, high level gene transfer to airway epithelial cells was achieved when virus was readministered to IL-12 treated animals at day 28, as seen in the day 31 results. The gamma-interferon treated animals were virtually indistinguishable from the animals treated with IL-12 in that efficient gene transfer was accomplished upon a second administration of virus. B . Immunological Assays - MLN Lymphocytes from MLN of the control group and IL-12 treated group of C57B1/6 mice harvested 28 days after administration of H5.010CBALP were restimulated in vitro with UV-inactivated H5.OlOCMVlacZ at 10 particles/ cell for 24 hours. Cell-free supernatants were assayed for the presence of IL-2 or IL-4 on HT-2 cells (an IL-2 or IL-4-dependent cell line) [Y. Yang et al, cited above] . Presence of IFN-7 in the same lymphocyte culture supernatant was measured on L929 cells as described [Y. Yang et al, cited above]. Stimulation index (S.I.) was calculated by dividing 3 H-thymidine cpm incorporated into HT-2 cells cultured in supernatants of lymphocytes restimulated with virus by those incorporated into HT-2 cells cultured in supernatants of lymphocytes incubated in antigen-free medium. The results are shown in Table 1 below. Table 1 3 H-Thymidine Incorporation fcpm±SD) IFN-7 liter Medium H5.OlOCMVlacZ S.I. (IU/ml) d C57B1/6 175 ± 40 2084 ± 66 11.91 80 anti-IL2 (1:5000) 523 ± 81 2.98 anti-IL4 (1:5000) 1545 + 33 8.83 C57B1/6 +IL12 247 ± 34 5203 ± 28 21.07 160 anti-IL2 (1:5000) 776 + 50 3.14 anti-IL4 (1:5000) 4608 ± 52 18.66 Stimulation of lymphocytes from regional lymph nodes with both recombinant adenoviruses led to secretion of cytokines specific for the activation of both T H1 (i.e., IL-2 and IFN-7) and T m (i.e., IL-4) subsets of T helper cells (Table 1) . Analysis of lymphocytes from the IL-12 treated animals stimulated in vitro with virus revealed an increased secretion of IL-2 and IFN-7 and a relative decreased production of IL-4 as compared to animals that did not receive IL-12 (i.e., ratio of IL-2/IL-4 was increased from 3 to 6 when IL-12 was used; Table 1) . C. Immunological Assays - BAL BAL samples obtained from animals 28 days after primary exposure to recombinant virus were evaluated for neutralizing antibodies to adenovirus and anti-adenovirus antibody isotypes as follows. The same four groups of C57B1/6 mice, i.e., control, CD4 + depleted, IL-12 treated and IFN-7 treated, were infected with H5.010CBALP. Neutralizing antibody was measured in serially diluted BAL samples (100 μl) which were mixed with H5.OlOCMVlacZ (1 x 10 6 pfu in 20 μl) , incubated for 1 hour at 37°C, and applied to 80% confluent Hela cells in 96 well plates (2 x 10 4 cells per well) . After 60 minutes of incubation at 37°C, 100 μl of DMEM containing 20% FBS was added to each well. Cells were fixed and stained for β-galactosidase expression the following day. All cells were lacZ positive in the absence of anti-adenoviral antibodies. Adenovirus-specific antibody isotype was determined in BAL by using enzyme-linked immunosorbent assay (ELISA) . Briefly, 96-well plates were coated with 100 μl of PBS containing 5 x lθ' particles of H5.OlOCMVlacZ for 18 hours at 4°C. The wells were washed 5 times with PBS. After blocking with 200 μl of 2% BSA in PBS, the plates were rinsed once with PBS and incubated with 1:10 diluted BAL samples for 90 minutes at 4°C. Thereafter, the wells were extensively washed and refilled with 100 μl of 1:1000 diluted alkaline phosphatase-conjugated anti-mouse IgG or IgA (Sigma) . The plates were incubated, subsequently washed 5 times, and 100 μl of the substrate solution (p-nitrophenyl phosphate, PNPP) was added to each well. Substrate conversion was stopped by the addition of 50 μl of 0.1M EDTA. Plates were read at 405 nm. The results are shown graphically in Figs. 11A through 11C, which summarize neutralizing antibody titer, and the relative amounts (OD^) of IgG and IgA present in BAL samples. The titer of neutralizing antibody for each sample was reported as the highest dilution with which less than 50% of cells stained blue. As demonstrated in the first bar of Figs. 11A through 11C, the cytokines identified in Table 1 above were associated in the control mice with the appearance of antibodies to adenovirus proteins in BAL of both the IgG and IgA isotypes that were capable of neutralizing the human Ad5 recombinant vector in an in vitro assay out to a 1:800 dilution. As shown in the second bar of the graphs of Figs. 11A through 11C, transient CD4 + cell depletion inhibited the formation of neutralizing antibody (Fig. 11A) and virus specific IgA antibody (Fig. 11C) by 80- fold, thereby allowing efficient gene transfer to occur following a second administration of virus. Fig. 11B shows a slight inhibition of IgG as well. More importantly, as shown in the third bar of the three graphs, IL-12 selectively blocked secretion of antigen specific IgA (Fig. 11C) , without significantly impacting on formation of IgG (Fig. 11B) . This was concurrent with a 32-fold reduction in neutralizing antibody (Fig. 11A) . The gamma-interferon treated animals (fourth bar of Figs. 11A through 11B) were virtually indistinguishable from the animals treated with IL-12 in that virus specific IgA (Fig. 11C) and neutralizing antibody (Fig. 11A) were decreased as compared to the control animals not treated with cytokine, but not to the extent obtained with those treated with IL-12. These studies demonstrate that inhibition of CD4 + function at the time of primary exposure to virus is sufficient to prevent the formation of blocking antibodies. The concordant reduction of neutralizing antibody with antiviral IgA suggests that immunoglobulin of the IgA subtype is primarily responsible for the blockade to gene transfer. All references recited above are incorporated herein by reference. Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention, such as selections of different modifications of adenovirus vectors selected to carry the VLDLR gene, or selection or dosage of the vectors or immune modulators are believed to be within the scope of the claims appended hereto. SEQUENCE LISTING (1) GENERAL INFORMATION! (i) APPLICANT! Truateea of Univeraity of Pennsylvania Wilson, James M. Kozara y, Karen F. Strauaa, Jerome F. (ii) TITLE OF INVENTION! Methods and Compoaitionβ for Gene Therapy for the Treatment of Defects in Lipoprotein Metabolism (iii) NUMBER OF SEQUENCES! 8 (iv) CORRESPONDENCE ADDRESS! (A) ADDRESSEE! Howson and Howson (B) STREET! Spring House Corporate Cntr., PO Box 457 (C) CITY! Spring House (D) STATE! Pennsylvania (E) COUNTRY! USA (F) ZIP! 19477 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE! Floppy disk (B) COMPUTER! IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0 Version #1.30 (vl) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER! (B) FILING DATE: (C) CLASSIFICATIONS (vii) PRIOR APPLICATION DATA! (λ) APPLICATION NUMBER! US 08/393,734 (B) FILING DATE: 24-FEB-1995 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Bak, Mary E. (B) REGISTRATION NUMBER: 31,215 (C) REFERENCE/DOCKET NUMBER: GNVPN009CIP1.PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 215-540-9200 (B) TELEFAX: 215-540-5818 (2) INFORMATION FOR SEQ ID NOil: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3656 base pairs (B) TYPE! nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) ( ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 392..3010 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CTCTGOGGGC CGCGGGTGCG GGTCGTOGCT ACCGGCTCTC TCCGTTCTGT GCTCTCTTCT 60 GCTCTCGGCT CCCCACCCCC TCTCCCTTCC CTCCTCTCCC CTTGCCTCCC CTCCTCTGCA 120 GCGCCTGCAT TATTTTCTGC CCGCAGCTCG βCTTGCACTG CTGCTGCAGC CCGGGGAGGT 180 GGCTGGGTGG GTGGGGAGGA GACTGTGCλλ GTTGTAGGGG AGGGGGTGCC CTCTTCTTCC 240 CCGCTCCCTT CCCCAGCCλλ GTGGTTCCCC TCCTTCTCCC CCTTTCCCCT CCCAGCCCCC 300 ACCTTCTTCC TCTTTCββλλ GGGCTGGTAλ CTTGTCGTGC GGAGCGAACG GCGGCGGCGG 360 CGGCGGCGGC GGCACCATCC AGGCGGGCAC C ATG GGC ACG TCC GCG CTC TGG 412 Met βly Thr Ser Ala Leu Trp 1 5 GCC GTC TGG CTG CTG CTC GCG CTG TGC TGG GCG CCC CGG GAG AGC GGC 460 Ala Val Trp Leu Leu Leu Ala Leu Cya Trp Ala Pro λrg Glu Ser Gly 10 15 20 GCC ACC GGA ACC GGG AGA λλλ GCC AAA TGT GAA CCC TCC CAA TTC CAG 508 Ala Thr Gly Thr Gly Arg Lya Ala Lya Cys Glu Pro Ser Gin Phe Gin 25 30 35 TGC ACλ AAT GGT CGC TGT ATT ACG CTG TTG TGG λλλ TGT GAT GGG GAT 556 Cys Thr λsn Gly λrg Cys lie Thr Leu Leu Trp Lys Cys λsp Gly λsp 40 45 50 55 GAA GλC TGT GTT GλC GGC λGT GAT GAA λλG λλC TGT GTλ AλG AλG ACG 604 Glu λsp Cys Val λap Gly Ser λsp Glu Lys λsn Cys Val Lys Lys Thr 60 65 70 TGT GCT GAA TCT GλC TTC GTG TGC λλC AAT GGC CAG TGT GTT CCC AGC 652 Cys λ a Glu Ser λap Phe Val Cys λsn λsn Gly Gin Cys Val Pro Ser 75 80 85 CGλ TGG λλG TGT GAT GGA GAT CCT GAC TGC GAA GAT GGT TCλ GAT GAA 700 λrg Trp Lys Cys λsp βly λsp Pro λβp Cys Glu λsp βly Ser λsp Glu 90 95 100 λGC CCA GAA CAG TGC CAT ATG AGA ACλ TGC CGC ATA CAT GAA ATC AGC 748 Ser Pro Glu βln Cys His Met λrg Thr Cys λrg lie His βlu lie Ser 105 110 115 TGT GGC GCC CAT TCT λCT CAG TGT ATC CCA GTG TCC TGG AGλ TβT GλT 796 Cys Gly λla His Ser Thr βln Cys lie Pro Val Ser Trp λrg Cys λsp 120 125 130 135 GGT GAA AAT GλT TGT GλC λCT GGA GAA GAT GAA GAA AAC TβT ββC AAT 844 βly βlu λsn λsp Cys λβp Ser βly βlu λsp βlu Glu λβn Cys Gly λsn 140 145 150 ATA λCλ TCT AGT CCC βλC GAG TTC ACC TGC TCC ACT GGC CGC TGC ATC 892 lie Thr Cys Ser Pro λβp Clu Phe Thr Cys Ser Ser Gly λrg Cys lie 155 160 165 TCC λββ λλC TTT GTλ TGC λλT GGC Cλβ βλT βλC TβC AGC GAT ββC λβT 940 Ser λrg λβn Phe Val Cyβ λβn βly βln λβp λβp Cyβ Ser λβp βly Ser 170 175 180 βλT βλβ CTβ βλC TβT GCC CCβ CCλ λCC TβT CGC GCC CλT βλβ TTC Cλβ 988 λβp βlu Leu λβp Cyβ λla Pro Pro Thr Cyβ βly λla Hie βlu Phe βln 185 190 195 TGC λβC λCC TCC TCC TGC ATC CCC λTC λβC TβG GTA TGC GAC βλT βλT 1036 Cyβ Ser Thr Ser Ser Cyβ lie Pro lie Ser Trp Val Cyβ λβp λβp λβp 200 205 210 215 GCλ GλC TGC TCC GλC Cλλ TCT βλT Gλβ TCC CTG βλβ Cλβ TGT ββC CGT 1084 λla λβp Cyβ Ser λβp Gin Ser λβp Glu Ser Leu βlu βln Cyβ βly λrg 220 225 230 Cλβ CCλ GTC ATλ CλC λCC AAG TGT CCλ GCC AGC GAA ATC CAG TGC GGC 1132 βln Pro Val He Hie Thr Lye Cyβ Pro λla Ser βlu He βln Cyβ βly 235 240 245 TCT GGC Gλβ TβC λTC CλT λλβ AAG TGG CCA TGT GλT GGG GλC CCT βλC 1180 Ser βly Glu Cyβ He Hie Lye Lye Trp λrg Cyβ λβp Gly λβp Pro λβp 250 255 260 TGC λλβ βλT ββC λβT GλT βλβ GTC λλC TGT CCC TCT Cβλ ACT TGC CGλ 1228 Cyβ Lye λβp Gly Ser λβp Glu Val λan Cyβ Pro Ser λrg Thr Cyβ λrg 265 270 275 CCT GλC Cλλ TTT GAA TGT GAG GλT GGC AGC TGC ATC CλT GβC λβC λβG 1276 Pro λβp Gin Phe Glu Cyβ Glu λβp βly Ser Cyβ He Hie βly Ser λrg 280 285 290 295 Cλβ TβT λλT GGT λTC Cβλ βλC TβT GTC GλT GGT TCC GλT GAA GTC λλC 1324 Cln Cyβ λβn Cly He λrg λβp Cyβ Val λβp βly Ser λβp βlu Val λβn 300 305 310 TCC λλλ λλT GTC λλT Cλβ TβC TTβ GβC CCT ββλ λλλ TTC λλβ TβC λβλ 1372 Cyβ Lye Aβn Val Λβn βln Cyβ Leu βly Pro βly Lye Phe Lye Cyβ λrg 315 320 325 λβT ββλ GAA TGC ATλ GAT ATC AGC λλλ GTλ TGT λλC CAG GAG CAG GλC 1420 Ser βly βlu Cyβ He λβp He Ser Lye Val Cyβ λβn Gin βlu βln λβp 330 335 340 TβC λββ βλC TGG AGT βλT βλβ CCC CTβ λλλ βλβ TβT CλT λTλ AAC GAA 1468 Cyβ λrg λβp Trp Ser λβp βlu Pro Leu Lye βlu Cyβ Hie He λβn Clu 345 350 355 TβC TTβ GTA λλT λλT GGT ββλ TGT TCT CλT λTC TβC λλλ βλC CTλ GTT 1516 Cyβ Leu Val λβn λβn βly βly Cyβ Ser Hie He Cyβ Lye λβp Leu Val 360 365 370 375 λTλ GGC TλC Gλβ TβT GλC TGT GCλ GCT Gββ TTT GAA CTβ λTλ βλT λGG 1564 He Gly Tyr Glu Cyβ λβp Cyβ λla λla Gly Phe Glu Leu He λβp λrg 380 385 390 λλλ λCC TGT Gβλ βλT ATT βλT GAA TGC Cλλ λλT CCλ GGA λTC TGC AGT 1612 Lye Thr Cyβ Gly λβp He λβp βlu Cyβ βln λβn Pro βly He Cys Ser 395 400 405 Cλλ λTT TβT λTC λλC TTλ λλλ GGC GOT TλC λλβ TβT GAA TβT λβT CGT 1660 βln He Cyβ He λβn Leu Lye βly βly Tyr Lye Cyβ βlu Cyβ Ser λrg 410 415 420 GCC TλT Cλλ λTβ βλT CTT βCT λCT GGC GTG TGC λλβ GCλ βTλ GGC λλλ 1708 λla Tyr βln Met λβp Leu λla Thr βly Val Cyβ Lye λla Val βly Lye 425 430 435 βλβ CCλ λβT CTβ λTC TTC λCT λλT Cβλ λβλ βλC λTC λββ λλβ λTT βGC 1756 βlu Pro Ser Leu He Phe Thr λen λrg λrg λβp He λrg Lye He βly 440 445 450 455 TTλ βλβ λββ λλλ βλλ TλT λTC Cλλ CTA βTT βλλ Cλβ CTA λβλ λλC λCT 1804 Leu Glu λrg Lye βlu Tyr He βln Leu Val βlu βln Leu λrg λβn Thr 460 465 470 GTG GCT CTC GλT GCT βλC λTT GCT GCC Cλβ λλλ CTA TTC TGG GCC GλT 1852 Val λla Leu λβp λla λβp He λla λla Gin Lya Leu Phe Trp λla Aβp 475 480 485 CTA λβC Cλλ λλG GCT λTC TTC λβT GCC TCλ ATT GAT βλC λλβ βTT GGT 1900 Leu Ser βln Lye λla He Phe Ser λla Ser lie λβp λβp Lye Val βly 490 495 500 AGλ CλT GTT λλλ ATG ATC GAC λλT GTC TλT λλT CCT GCλ GCC ATT GCT 1948 λrg Hie Val Lya Met He λβp λβn Val Tyr λβn Pro λla λla He λla 505 510 515 βTT βλT TGG GTG TλC λλG λCC λTC TλC TGG λCT GλT GCG GCT TCT λλG 1996 Val λep Trp Val Tyr Lye Thr He Tyr Trp Thr λβp λla λla Ser Lye 520 525 530 535 ACT ATT TCA βTλ GCT λCC CTA GλT ββλ λCC λλβ λββ λλβ TTC CTβ TTT 2044 Thr He Ser Val λla Thr Leu λep βly Thr Lye λrg Lye Phe Leu Phe 540 545 550 λλC TCT CλC TTC CGλ βλβ CCT GCC TCC λTλ GCT CTβ βλC CCλ CTG TCT 2092 λen Ser Aβp Leu λrg Glu Pro λla Ser He λla Val Aβp Pro Leu Ser 555 560 565 GβC TTT CTT TλC TGG TCλ GλC TGG GGT GAA CCλ GCT λλλ λTλ Cλλ λλλ 2140 βly Phe Val Tyr Trp Ser λβp Trp βly βlu Pro λla Lye He βlu Lye 570 575 580 GCΛ GGA ATG λλT ββλ TTC βλT λβλ CGT CCλ CTG CTβ λCλ GCG GλT λTC 2188 λla Gly Met λβn βly Phe λβp λrg λrg Pro Leu Val Thr λla λβp He 585 590 595 Cλβ TGG CCT ΛΛC Gβλ ATT ACΛ CTT βλC CTT ATA λλλ λβT CGC CTC TλT 2236 Gin Trp Pro λen Gly He Thr Leu λβp Leu He Lye Ser λrg Leu Tyr 600 605 610 615 TGG CTT GλT TCT λλβ TTβ CλC λTC TTλ TCC AGC GTG GλC TTG λλT ββC 2284 Trp Leu Λβp Ser Lye Leu Hie Met Leu Ser Ser Val λβp Leu λβn Cly 620 625 630 Cλλ CλT CCT λββ λTλ βTλ CTA λλβ TCT CTβ GAG TTC CTA GCT CλT CCT 2332 Gin λβp λrg λrg He Val Leu Lye Ser Leu Glu Phe Leu λla Hie Pro 635 640 645 CTT GCλ CTA λCλ ATλ TTT GAG GλT CGT GTC TλC TGG ATA GΛT GGG GAA 2380 Leu Ala Leu Thr He Phe Glu λβp λrg Val Tyr Trp He λβp Gly Glu 650 655 660 λλT βλλ GCλ GTC TλT ββT CCC λλT λλλ TTC λCT GGA TCλ βλβ CλT GCC 2428 λβn βlu λla Val Tyr βly λla λβn Lye Phe Thr βly Ser βlu Hie λla 665 670 675 λCT CTλ CTC λλC λλC CTβ λλT GλT βCC Cλλ βλC λTC λTT GTC TλT CλT 2476 Thr Leu Val λβn λβn Leu λβn λβp λla Gin λep He He Val Tyr Hia 680 685 690 695 βλλ CTT GTλ Cλβ CCλ TCλ GGT λλλ λλT TGβ TGT βλλ βλλ CλC λTC βλβ 2524 βlu Leu Val βln Pro Ser βly Lye λβn Trp Cyβ βlu βlu λβp Met βlu 700 705 710 λλT ββλ ββλ TβT βλλ TλC CTλ TβC CTβ CCλ OCA CCλ CAG λTT λλT βλT 2572 λβn βly βly Cyβ βlu Tyr Leu Cyβ Leu Pro λla Pro βln He λen λβp 715 720 725 CλC TCT CCλ λλλ TλT λCC TGT TCC TGT CCC λGT GGG TλC λλT GTλ βλβ 2620 Hie Ser Pro Lye Tyr Thr Cyβ Ser Cyβ Pro Ser Cly Tyr λβn Val βlu 730 735 740 βλλ λλT βGC Cβλ βλC TβT Cλλ λβT λCT GCΛ ACT ACT GTG ACT TλC λβT 2668 βlu λβn βly λrg λβp Cyβ Cln Ser Thr λla Thr Thr Val Thr Tyr Ser 745 750 755 βλβ λCλ λλλ CλT λCC λλC λCA λCλ GAA ATT TCλ GCA ACT AGT Gβλ CTλ 2716 βlu Thr Lye λβp Thr λβn Thr Thr βlu He Ser λla Thr Ser βly Leu 760 765 770 775 βTT CCT ββλ GGG λTC λλT GTG λCC λCλ GCA GTλ TCλ GAG GTC AGT GTT 2764 Val Pro Gly Gly He λβn Val Thr Thr λla Val Ser Glu Val Ser Val 780 785 790 CCC CCλ λλλ Gββ ACT TCT GCC GCA TGβ GCC ATT CTT CCT CTC TTG CTC 2812 Pro Pro Lye Gly Thr Ser Ala λla Trp λla He Leu Pro Leu Leu Leu 795 800 805 TTλ GTG λTβ GCA GCλ GTλ GGT GGC TλC TTG ATG TGβ CGG λλT TGG Cλλ 2860 Leu Val Met λla λla Val Gly Gly Tyr Leu Met Trp λrg λβn Trp Gin 810 815 820 CλC λλβ λλC λTβ λλλ AGC ATG λλC TTT βλC λλT CCT CTβ TλC TTβ AAA 2908 Hie Lye λen Met Lye Ser Met λen Phe λβp λβn Pro Val Tyr Leu Lye 825 830 835 λCC ΛCT βλλ βλβ βλC CTC TCC λTλ βλC λTT GGT λβλ CλC λβT GCT TCT 2956 Thr Thr βlu βlu λβp Leu Ser He λβp He βly λrg Hie Ser λla Ser 840 845 850 855 βTT ββλ CλC λCC TλC CCλ GCA λTλ TCλ GTT GTλ AGC ACλ GλT GλT GλT 3004 Val Gly Hie Thr Tyr Pro λla He Ser Val Val Ser Thr λep λβp λβp 860 865 870 CTλ GCT TβλCTTCTCT GACAAATGTT GACCTTTGAG GTCTAAACAA ATAATACCCC 3060 Leu λla CGTCGGAATG GTλλCCβλβC CλGCλβCTCλ AGTCTCTTTT TCTTCCTCTC CGCTGGλλGΛ 3120 ΛCATCAAGAT λCCTTTGCGT GGΛTCλλGCT TβCTβTλCTT βACCβTTTTT ATATTACTTT 3180 TβTAλλTλTT CTTCTCCλCλ TTCTλCTTCλ CCTTTGGλTG TGGTTλCCGλ GTATCTGTAA 3240 CCCTTGAATT TCTAGACAGT ATTGCCACCT CTGGCCAAAT ATGCACTTTC CCTAGAAAGC 3300 CλTλTTCCλβ CλβTOλλλCT TOTOCTλTλG TβTλTλCCλC CTβTλCλTλC λTTβTλTλββ 3360 CCλTCTβTλλ λTλTCCCλβλ βλλCλλTCλC TλTTCTTλλβ CλCTTTβλλλ λTλTTTCTλT 3420 βTλλλTTλTT βTλλλCTTTT TCλλTββTTβ ββλCλλTββC λλTλGGλCλλ λλCGGGTTAC 3480 TλλβλTGλλλ TTβCCλλλλλ λλTTTλTλλλ CTλλTTTTββ TλCβTλTGλλ TGATATCTTT 3540 GλCCTCλλTC GλββTTTβCλ λλβλCTβλβT βTTCλλλCTλ CTβTλCλTTT TTTTTCλλβT 3600 βCTλλλλλλT TλλλCCλλβC λβCTTλλλλλ λλλλλλλλλλ λλλλλλλλλλ λλλλλλ 3656 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 873 amino acidβ (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met βly Thr Ser λla Leu Trp λla Val Trp Leu Leu Leu λla Leu Cye 1 5 10 15 Trp λla Pro λrg Clu Ser βly λla Thr βly Thr βly λrg Lye λla Lye 20 25 30 Cye βlu Pro Ser βln Phe βln Cyβ Thr λβn Cly λrg Cyβ He Thr Leu 35 40 45 Leu Trp Lye Cyβ λep βly Aβp βlu λβp Cyβ Val λβp Cly Ser Aβp βlu 50 55 60 Lye λen Cye Val Lye Lye Thr Cye λla Clu Ser λβp Phe Val Cye λβn 65 70 75 80 λen βly βln Cye Val Pro Ser λrg Trp Lye Cyβ λep Cly λep Pro λep 85 90 95 Cye βlu λep βly Ser λβp βlu Ser Pro βlu βln Cye Hie Met λrg Thr 100 105 110 Cyβ λrg He Hie Clu He Ser Cye Cly λla Hie Ser Thr βln Cye He 115 120 125 Pro Val Ser Trp λrg Cye λep Cly βlu λβn λβp Cyβ λβp Ser Cly βlu 130 135 140 λβp βlu βlu λβn Cye Cly λβn He Thr Cyβ Ser Pro λβp Clu Phe Thr 145 150 155 160 Cye Ser Ser Cly λrg Cye He Ser λrg λβn Phe Val Cye λβn Cly βln 165 170 175 λβp λβp Cyβ Ser λβp Cly Ser λβp βlu Leu λβp Cyβ λla Pro Pro Thr 180 185 190 Cye Cly λla Hie βlu Phe βln Cye Ser Thr Ser Ser Cye He Pro He 195 200 205 Ser Trp Val Cyβ λβp λβp λβp λla λβp Cyβ Ser λβp Cln Ser λβp βlu 210 215 220 Ser Leu βlu βln Cyβ βly λrg βln Pro Val He Hie Thr Lye Cye Pro 225 230 235 240 λla Ser βlu He βln Cye βly Ser βly βlu Cyβ He Hie Lye Lye Trp 245 250 255 λrg Cye λβp Cly λβp Pro λβp Cyβ Lye λep Cly Ser λβp βlu Val λan 260 265 270 Cyβ Pro Ser λrg Thr Cye λrg Pro λβp Cln Phe βlu Cye βlu λβp βly 275 280 285 Ser Cyβ He Hie βly Ser λrg βln Cye λβn Cly He λrg λβp Cyβ Val 290 295 300 λβp Cly Ser λβp βlu Val λan Cyβ Lye λen Val λen Cln Cyβ Leu βly 305 310 315 320 Pro βly Lye Phe Lye Cye λrg Ser βly βlu Cyβ He λβp He Ser Lye 325 330 335 Val Cyβ λβn βln βlu βln λβp Cyβ λrg λβp Trp Ser λβp Clu Pro Leu 340 345 350 Lye βlu Cye Hie He λβn Clu Cyβ Leu Val λβn λβn Cly βly Cyβ Ser 355 360 365 Hie He Cyβ Lye λβp Leu Val He Cly Tyr βlu Cye λβp Cyβ λla λla 370 375 380 Cly Phe βlu Leu He λβp λrg Lye Thr Cyβ Cly λβp He λβp βlu Cyβ 385 390 395 400 βln λβn Pro βly He Cyβ Ser Cln He Cyβ He λβn Leu Lye Cly βly 405 410 415 Tyr Lye Cyβ βlu Cyβ Ser λrg λla Tyr βln Met λep Leu λla Thr βly 420 425 430 Val Cyβ Lye λla Val Cly Lye βlu Pro Ser Leu He Phe Thr λβn λrg 435 440 445 λrg λβp He λrg Lye He βly Leu βlu λrg Lye βlu Tyr He βln Leu 450 455 460 Val βlu βln Leu λrg λen Thr Val λla Leu λβp λla λβp He λla λla 465 470 475 480 βln Lye Leu Phe Trp λla Aβp Leu Ser βln Lye λla He Phe Ser λla 485 490 495 Ser He λβp λβp Lye Val βly λrg Hie Val Lye Met He λβp λβn Val 500 505 510 Tyr λβn Pro λla λla He λla Val λβp Trp Val Tyr Lye Thr He Tyr 515 520 525 Trp Thr λβp λla λla Ser Lye Thr He Ser Val λla Thr Leu λep βly 530 535 540 Thr Lye λrg Lye Phe Leu Phe λen Ser λβp Leu λrg βlu Pro λla Ser 545 550 555 560 He λla Val λβp Pro Leu Ser βly Phe Val Tyr Trp Ser λβp Trp βly 565 570 575 βlu Pro λla Lye He βlu Lye λla βly Met λβn βly Phe λβp λrg λrg 580 585 590 Pro Leu Val Thr λla λep He βln Trp Pro λen βly He Thr Leu λep 595 600 605 Leu He Lye Ser λrg Leu Tyr Trp Leu λep Ser Lye Leu Hie Met Leu 610 615 620 Ser Ser Val λep Leu λen βly βln λep λrg λrg He Val Leu Lye Ser 625 630 635 640 Leu βlu Phe Leu λla Hie Pro Leu λla Leu Thr He Phe βlu λβp λrg 645 650 655 Val Tyr Trp He λep βly βlu λβn βlu λla Val Tyr βly λla λβn Lye 660 665 670 Phe Thr Gly Ser βlu Hie λla Thr Leu Val λen λen Leu λβn λβp λla 675 680 685 βln λep He He Val Tyr Hie βlu Leu Val βln Pro Ser βly Lya λβn 690 695 700 Trp Cyβ βlu βlu λep Met βlu λβn βly βly Cyβ Clu Tyr Leu Cye Leu 705 710 715 720 Pro λla Pro βln He λβn λβp Hie Ser Pro Lye Tyr Thr Cyβ Ser Cye 725 730 735 Pro Ser Gly Tyr λβn Val βlu βlu λβn βly λrg λep Cye βln Ser Thr 740 745 750 λla Thr Thr Val Thr Tyr Ser βlu Thr Lye λep Thr λβn Thr Thr βlu 755 760 765 He Ser λla Thr Ser βly Leu Val Pro βly βly He λβn Val Thr Thr 770 775 780 λla Val Ser βlu Val Ser Val Pro Pro Lye βly Thr Ser λla λla Trp 785 790 795 800 λla He Leu Pro Leu Leu Leu Leu Val Met λla λla Val βly βly Tyr 805 810 815 Leu Met Trp λrg λβn Trp βln Hie Lye λβn Met Lye Ser Met λβn Phe 820 825 830 λβp λβn Pro Val Tyr Leu Lye Thr Thr Glu Glu λβp Leu Ser He λβp 835 840 845 He βly λrg Hie Ser λla Ser Val βly Hia Thr Tyr Pro λla He Ser 850 855 860 Val Val Ser Thr λβp λβp λβp Leu λla 865 870 (2) INFORMλTION FOR SEQ ID NO:3: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 9592 baβe pairβ (B) TYPE: nucleic acid (C) STRλNDEDNBSS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GλλTTCβCTλ GCATCλTCAA TλλTλTλCCT TλTTTTββλT TβλλβCCλλT λTβλTλλTβλ 60 GGGGGTGGλG TTTGTGλCGT GGCGCGGGGC βTβββλλCββ GGCGGGTGAC GTAGTAGTGT 120 ββCββλλβTβ TβλTβTTβCλ λGTGTGGCGG AΛCACATGTA AGCGACGGAT GTββCλλλλβ 180 TGACGTTTTT GGTGTGCGCC GGTGTACACA ββλλβTGλCλ ATTTTCGCGC GβTTTTλββC 240 GGλTGTTGTλ GTAAATTTGG GCGTAACCGA GTλλβλTTTβ GCCλTTTTCβ CβββλλλλCT 300 GλATAAGAGG AAGTGAAATC TGAATAATTT TβTβTTλCTC ATAGCGCGTA ATATTTGTCT 360 λGGGAGATCA GCCTGCλGGT CGTTACATΛA CTTλCGGTAA ATGβCCCCCC TGGCTβλCCC 420 CCCλλCCλCC CCCGCCCATT βλCCTCλλTλ ATGACGTATG TTCCCλTλβT AACGCCAATA 480 GββλCTTTCC ATTGACGTCA ATGGGTGβλβ TλTTTACGGT AλλCTGCCCλ CTTGGCAGTA 540 CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TλλλTGGCCC 600 GCCTGβCλTT ATGCCCAGTλ CATGACCTTA TGGGλCTTTC CTλCTTGβCλ GTACATCTλC 660 GTATTAGTCA TCGCTλTTλC CλTββTβλTβ CGCTTTTGGC AGTACATCAA TGGGCGTGGA 720 TλβCββTTTβ ACTCACGGGG ATTTCCAAGT CTCCλCCCCλ TTGACGTCAA TGGGAGTTTG 780 TTTTGGCACC λλλλTCλλCβ ββλCTTTCCλ λλλTβTCβTλ λCλλCTCCGC CCCλTTGλCG 840 CλλλTGGGCC GTAGGCGTGT λCββTβββλβ βTCTλTλTλλ βCλCλβCTCT CTGGCTλλCT 900 λGλβλλCCCλ CTGCTTλλCT GβCTTATCβλ AλTTλλTλCG ACTCACTATA GββλGλCCCλ 960 AGCTTCTCTG CβGβCCβCββ GTβCβGGTCβ TCGCTλCCββ CTCTCTCCβT TCTβTGCTCT 1020 CTTCTGCTCT CGGCTCCCCλ CCCCCTCTCC CTTCCCTCCT CTCCCCTTGC CTCCCCTCCT 1080 CTGCAGCGCC TGCATTATTT TCTGCCCβCλ GCTCGGCTTC CλCTGCTGCT GCλβCCCβGG 1140 GλββTββCTβ GβTβGGTβββ GAGGAGACTG TGCAAGTTGT λGGGGλGGββ GTGCCCTCTT 1200 CTTCCCCGCT CCCTTCCCCλ GCCλλGTGGT TCCCCTCCTT CTCCCCCTTT CCCCTCCCAG 1260 OZXε V99XW9XW 99YD9YYYYY 9VXWWX39 V33W9X9999X V9V3X99 X3VXXX9XXX 090ε 099X3X9X3Y 333V99X9X39VXV33X339 X339V9V939 XX3V9X3X3V VXXX9X33XX OOOC OW99V9W33W99XV9VX 333VX39VX9 V3XXXVX3V9 WX3XX39939XV9X3V99X 0*63 3VX3XV33V9 W3VX9X999 XXV9XX9X39 XXV339V39X 33XWXVX3X 9XW3V93XV 088Z 9XWWXX9X V3V9VX99XX 99W3V9XV9 XXW3X339X 9V3XX3XVX399WW339V 0Z8Z VX3XV933999X3XXVX3W V9V3339X39 XXV3V9X39X V93X3X399X 9X3V3VW9V 09LZ VX39V3W9X X9VX3W33X VXVXW9WV 99V9V9VXX399XXV9W99 V3XV3V9V9V 004.Z V93XWX3V3 XX3XV9X3X9 W339V9YW 399VX9V390 W39X9X9399X3VX39XX3 0*92 XV99XVW3X VX339X93X9 VX9XW9X9X 9W3VXX99399WWXX3V V3XVX9XXXV 08SZ W3X9V39X3 XW99V33XV VW339XW9 XV9XXVXV9V 99X9X33VW V99VXV9VXV 0392 9X3W9XXX999X39V39X9 X3V9X9X9V93VX399VXVX X9VX33V9W V39X3XVXV3 09*Z X3XX9XV99X 99XWXVWX 99XX39XW93WVXVXV3X 9X9V9VW9X 33339V9XV9 OOfrZ X9V99X3V999V39X3V99V 39V99V33W X9XVX9VW39V3XVXV9VX V39XW9V99 Ofr€Z X9W9V39X9 W3XXWW99X333999XX 39X9V3XW3 X9XVWW39 X3W3X9W9 0823 XV933XX99X V93X9X9X3V 9V933XVX99 XWX9X9V399V39V399XV 33XV39X39V OZZZ 399XV99V9X 9XW9XXXW 30V9X33V9339XX3W93X 3X333X9X3V V3X99V9XV9 091Z X9V399XV99 W39X3V9X333V9999XV9 X9XV9399X9 W9WXV33X V39X9V9399 001Z X3X39939X9 V33XVW0O9 V339V33X9X 9W33V3V3V XV3X9V339V 3X93399X9X 0»0Z 9V39V99X333X9V9XV9X3 XW33V903X 39X3V9V39X V9XV93V939 XVX999X39V 0861 3XV3333XV39X33X33X33 V39V39X9V33XX9V9XV3339399X9X33 W33933339 0Z6X X9X3V99X39 V9XV9X9V399XV939V39X 3V9XV99V3399XW39XVX 9XXX3W99V 0981 33X3XV39X393399X9V33 X39X33V3XX 9V93V9333X 9VX9XV3WX VXW399X9X 0081 3WW9W9X V9W9V99X9 V3V9X9XXV9 XWW9X99X V9X9XV9V99 X33X9X9V33 0»£I 3XVX9X9V3X 3VX3XXV3339399X9X39V 3XVW9XV3V XV39339XV3 W9V9XVXV3 0891 39X9V3W9V 3339VW9XV 9V3XX99XV9 W909X3V9X 33XV9V99XV 9X0X9W99X 0Z9X V9339V333X X9X9X9V3399XW3W39X 9X93XX3V9X 3XW9X39X9 X93V9W9W 099X VX9X9X3W9 WW9XV0X9 V3993V9XX9 X9X3V9W9X V9999XV9X9 XWV99X9XX OOSX 0X390VXXVX 9X390X99XV W3V39X9V33XXW333X333W9X9XW V339VWV9V 0»»I 99933W9933V33939939 V9V999333390990X39X9 X39393X39X 39X399X3X9 Oβ i 33999X3X393933X93V3999XV33V3999399V33XV33V399399399399399399 OZCX 3993993W939V9939X93 X9XX3WX99 X3999W993 XXX3X33XX3 XX33V33333 OL ltO€0/96Sn/I3d 98Z9Z/96 OΛ\ OΘ6fr 3933XV3XX9333XX39V39 X9Y39YY39X X393339V9X 33XXX39XXX 3V9X3V9X9X 0Z6* XV99939333933V339V39 X39339V3XX 0933933933 X339V39X3V 9V99XX9339 098» 3W99X3X9X 933V9V93VX 33V9XX33VX 3VX3X3VW393339X33X9333393X99X OOβfr V9XXV39V33 X3999XV9X9 XW9V3X939 X999933999 XV333339XV 39393W3V9 OtLV XXXVXV3X39 V9X9XXV39V V99XV9XXX93X3W33V39 V9XV339339339339V39V 089* 39XXXX9X3X VX9XXXX9VX 9XVXX3X99999X99YVXYX VXYV9YYY999X999YYXX3 0Z9* 99X93999X9 X9XVYY9X3V X99Y9XXY9Y 3YXY9YV9XY 939YX3X399 XXX9V9X393 09S» 9333Y39X3399X39X99XX 3V3XY933399V9X39Y99V 933Y9X9XY99X39XY9X9X 00S» 339V33VY99 YXXVXV3YW X99390X9X9 Y939X333Y9 V39X99Y33V 39333Y9Y9X 0*fr* Y93YX99Y9X 39X99YY99X 3XY9X399Y39X33V93X9Y 9YX3X33XY990XXXXXX09 OΘC* Y999X9X99V 99999Y3XX99Y3XXX9XVX XXXV3XXY39 XXYY3YV3VY 3YVXX9YV3Y OZ fr YYXYY39X39 YYXYXXY33Y VX9XXXYXXX 39XXYX39XY 9X9XXXYYV9 X9XXXYXXX3 09Z* 9XYYYYYW9 X9Y39XYY9Y X3YY3Y33VY Y3Y99XXX9V 9XY9XXY3VX Y9YVXY9XY3 OOZfr Y9Y33XY9999393399399 X3Y3V3XY33 XYXY9Y39X3 XXYY99X39X 9X9Y339339 Ofrlfr 93YYX9YX3Y 33XV993X39 Y933YX99XX 39VY3XY99X 939XXX33YX Y9W3XY3VY 080fr 9YV99X3993 X3X33XX3XX XXX3X3X9YV 9X39Y39V33 9Y933VYX99 XYY993X933 0Z0» 333VXYYXVY V3YWX3X99 Y9XXX33V9X X9XYYY3Y9X 9X3XX3Y9XX 39YX3XY9XV 096ε 9XY9Y3V39Y YX9XX9Y3XY XYY39Y333Y X93Y3Y3Y99 XX9X3XX39X 9Y3Y3Y9YX9 006ε 9XXY3Y9VXY 33X3X33V99 Y9YV9X3Y33 YYVY9XX3VX 9X9X33XYY3 V9XXX3YV9X Ofβ Y39WYY9XY 3VY9YY3V3Y Y399XXYY99399X9XV9XX 3YX399X99Y X9V39Y399X 09 L£ Y9X9YXX3X39XX3X3X33X X3XXV33999 XY39039X3X X3Y999YYYV 33333XX9X9 ZLZ Y3X99Y9V3X YX9V39Y3V33Y9X9XYY3X Y999Y99X33 XX9YX3V99X 9YX3VY39Y3 099ε XXXYVY9Y3V Y3Y3YY93YX V9YYYY3V9Y 9YXX3Y9X9X 3YX3VY39X3 VX9YW3X9X 009 3V0V93399X YVYY99Y9VX 9XYV3YX999 X9Y333X9X33XX9X33YXV XYVYV33X3X 0»S 3Y3XV9XYVX XY9Y3Y33Y39Y339X339X YX33YXYV9X 9XY99V99XY V9Y99XY3Y9 08*ε YV9YV9X9X99XXYYYYYX99Y3XY339Y3 YX9XX3YY9X Y3XYX3X9XX Y3XY3Y0YY3 OZfrε 339XY9XYY9 X33YY3YY3X 9YX3X3Y339 XY39V9Y3XV 99X3Y3XXYV YXYY339X99 09εε XVX3X9Y39V Y9XVYYY9999XY9VXY90X 3YX3X9X93X V99Y9XXXVX YY3YVX3Y39 OO XX3X33XY3X 39VX33XX9Y 99X3X3X9YV YX3VX9YXY99YX93XY9YV 3399XYV9XX OVZ 3Y99X939Y33XYXX9XY3Y 39XX9VYX3X XY9XX399XX YX3X3393X9 VYYYVXYXX3 081ε 3Y9XX3Y3YX XYY993VYX3399X9Y33XV XY9939Y3Y9 X99X3Y33X93Y9YXV93XX XL IWεθ/96Sfl/JOd 9W9Z/960M 0*89 9X399WW93V9X993X39 3V33X9X993 39V9XV33XX X99X3X33VX X3XXX939XV 0SL9 9XXXXX39XV 33333XXX99 V33VWW3X 99993XX0O3 99X3X39V9X 99V339V93V QZL9 33XXV393X3 X993V9V39333399V3933 93933XV399 VX9V999933 XXV933VXW 0999 V9V9393999 XX39V9VX93 999V9XXXX3 V9V39X9V39 999V93V393 39099V99V9 0099 9XX3339XX3 9V3939399X X33399X93990933X3333 9V33X9VXV3 X9X99XV33V 0frS9 9XXXV39VX99V33993X93 939X33303X X3X99339X3 909W9X39X 99X39X33X9 08»9 9X399V9XX3900X999V3399X390909X 399Θ03X390 9X9999W9X 993V3X999X 0Z»9 3X9VX939V3 X93X33X999 V3939990V33XXX3X9XV3 X999V339993V9V33X93X 09£9 39X993X9VX 9V3993VX9X 393XXX3093 9999XX9993 93XXX93X33 X3XVXV39V3 00 9 3XV93X3XV3 993VX3X39X 33V3X993X3 9V3V333X99399V33XX9V 39W33V9XX 0tZ9 X939V9XXXX 39XV399VX933933X903V 9V9XXX993V V3XXXXX9W V39W99W3 08X9 9XX3XX9V39 VXV939V333 93393X39399W9V33903 XVW33V9X3 33XXXX9XV3 0ZX9 93X3V9X333 X9XV39WXX 90XX3V33999999V39V9X 333XV3X933 9X39V39X39 0909 V9V9WXX9V X99X3W39X 99933VXXVX 33V3V3XVW X9033999X9 9339V3933V 0009 XX3V939X39 V39V9X33XX 99V39VW9V V999X39V3X V9V9999VX9 99933XXX99 0»6S 3WW9W9X V93999939X 33VX3X9XV3 XV999999XV 9V3XX9V9XX X390V333XX 088S XV39XXXV9V 3V3X333VXX 9VX939999V 3339933XV33XX99XWXV X9939X3V9V 0Z8S 339X999V9939993909W V3VXXXXXV3399VXV3X93 XV9V9XV99V 33XX9X9XX9 09 LS VXV3XOOWX 3V3XV990X3 XXXVXV9W93090X039009939993V33 3999XW399 00LS XV9XWXV33 X90XXV39XV 33XXXXV9W 33X33V9X9X X33390V9V99XX3W9W9 0»9S 9X939XVW99W9VXX39V X9XV3X9XXX VW999XX3V 39X9933XVX 9X9V3V39V3 08S 3V33W9V39 X9XX9XV3XX V999933X333XVXV339V3 33XX9XVX39 9XX99VXXXX OZSS XVX9X3V99X X3XV39XV9V 9XVXV9999X 93VXV39X99 9XV999X39V VXX9909VW 09»S 3VXXX9X9W X9X99XX33390V39999V3 39XXV9X39V V39VX9V3XX X3X9XWWV 00»S X339X99X93 999X3909V99V39VX93X9 V33XV9XV9V X9XX9X99X999939X39XV 0» S 3XX39V9V39 X3V33V39VX 99V99X9999 X3X3X93339 WXV3999XV 3VXV9V3XX9 08ZS XV99X3X3V9 X0ΘVWX99X 03V99V33XX XXXXVX9X9X 33X990V9XX 93X993X3X9 OZZS 939V33V99933399VX993 0O93939XXX X9999VXXXV XXX3XOX39X X3X9X9W39 09XS W3XV99XXX V90XXX9X3X 3V0V33WW WXWVXV3V WVXXX9909 XW333X333 OOXS 3X33XX399V V9X3339X3X XX99V39V33939X3XY99X X9X39V39V3 X3XXX0OX9X 0»0S WXX3W999333V9XXX3X XV99XXW3V 399XXXX3X3 993V9XX9W 3V9XV90903 ZL lH)εθ/96SO/XDd 98Z9Z/96 O A TCCOTCTCCC CβTλTλCλOλ CTTβλβλβOC CTGTCCTCGA CCGATGCCCT TβλβλβCCTT 6900 CλλCCCλβTC λβCTCCTTCC ββTβββCβCβ GGGCλTGλCT ATCGTCCCCG CλCTTλTβλC 6960 TβTCTTCTTT λTCATβCλλC TCCTλGGλCA ββTGCCβGCλ GCGCTCTGGG TCλTTTTCGG 7020 CβλββλCCβC TTTCGCTGGA GCGCGλCGλT βλTCββCCTβ TCGCTTGCGG TATTCGGAAT 7080 CTTGCλCGCC CTCGCTCλλβ CCTTCGTCAC TCGTCCCGCC λCCλλλCGTT TCGβCβλβλλ 7140 βCλGGCCATT λTCGCCGGCλ TGβCGGCCGA CGCGCTGGGC TλCβTCTTβC TββCGTTCβC 7200 βλCβCβλββC TββλTββCCT TCCCCλTTλT CλTTCTTCTC GCTTCCGGCG GCλTCGGGλT 7260 GCCCGCGTTG CλββCCλTCC TGTCCλGGCλ GβTλβλTβλC GλCCλTCλββ GACAGCTTCA 7320 λββλTCβCTC βCβGCTCTTλ CCλβCCTλλC TTCGλTCλCT GGACCGCTGA TCβTCACCβC 7380 βλTTTλTβCC GCCTCGGCGA GCλCλTββλλ CGGGTTGGCA TGGATTGTAG GCGCCGCCCT 7440 λTλCCTTGTC TGCCTCCCCβ CGTTGCGTCG CββTβCλTββ λβCCβββCCλ CCTCβλCCTG 7500 λλTGβλλβCC ββCββCλCCT CβCTλλCββλ TTCλCCλCTC CλλβλλTTββ λβCCλλTCλλ 7560 TTCTTGCGGA βλλCTβTβλλ TGCβCλλλCC λλCCCTTββC λGλλCλTλTC CATCGCGTCC 7620 GCCATCTCCA βCACCCβCλC GCββCβCATC TCGGGCλGCG TTGGGTCCTβ GCCACGGGTG 7680 CGCATGATCG TβCTCCTCTC GTTCλββλCC CGβCTλββCT GGCGGββTTβ CCTTACTGGT 7740 TAGCλβλλTβ AATCλCCβλT λCGCβλβCβλ ACGTGλλβCG λCTGCTβCTβ CλλλλCGTCT 7800 GCβλCCTGλG CλλCλλCλTG AATGGTCTTC GGTTTCCGTG TTTCGTAAAG TCTGβλλλCC 7860 CββλλβTCλβ CGCCCTGCAC CATTATGTTC CββλTCTβCλ TCGCλββλTβ CTGCTGGCTλ 7920 CCCTGTGGλλ CACCTACATC TGTλTTλλCβ AλβCCTTTCT CλλTGCTCAC GCTGTAGβTλ 7980 TCTCAGTTCC GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACβλλC CCCCCGTTCλ 8040 GCCCGACCGC TGCGCCTTλT CCGGTAACTA TCGTCTTGAG TCCλλCCCGβ TλλGλCλCGλ 8100 CTTATCGCCλ CTGGCAGCAG CCλCTGGTλλ CAGGATTAGC AGAGCGAGGT ATGTλGGCβ 8160 TβCTACλβλβ TTCTTGAAGT GGTGGCCTAA CTλCββCTλC ACTAGAAGGA CλGTATTTGG 8220 TλTCTGCβCT CTGCTβλλGC CAGTTACCTT CGβλλλλλβλ GTTββTλβCT CTTβλTCCGG 8280 CλλλCλλλCC ACCβCTGGTλ GCGGTGGTTT TTTTCTTTβC λλGCλGCλβλ TTλCGCβCλβ 8340 λλλλλλλGGλ TCTCAAGAAG ATCCTTTGAT CTTTTCTλCβ GββTCTβλCC CTCAGTGGAA 8400 CGλλλλCTCλ CGTTAAGGGA TTTTGβTCAT GAGATTATCA AAAAGGATCT TCλCCTλGλT 8460 CCTTTTλλλT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT λλλCTTGGTC 8520 TGACAGTTAC CλλTGCTTλλ TCλCTβλββC λCCTλTCTCλ GCGATCTCTC TλTTTCGTTC 8580 ATCCATλGTT GCCTCλCTCC CCGTCCTβTλ GATAACTACG ATλCGGGλGG GCTTACCλTC 8640 TGGCCCCAGT GCTGCAATGA TλCCGCGλGλ CCCλCGCTCλ CCGGCTCCAG ATTTATCAGC 8700 λλTλλλCCλβ CCλGCCββλλ ββOCCβλβCβ CλGAAGTGGT CCTβCλλCTT TλTCCGCCTC 8760 CλTCCλCTCT λTTλλTTOTT βCCOOOλλβC TλGλGTλλOT λβTTCβCCλβ TTλATAGTTT 8820 βCGCλλCGTT GTTGCCλTTC CTCCλββCλT COTβGTGTCA CβCTCGTCGT TTββTλTββC 8880 TTCλTTCλβC TCCOGTTCCC λλCβλTCλλβ βCβλβTTλCλ TβλTCCCCCλ TGTTGTGCAA 8940 λλλλβCββTT λGCTCCTTCC GTCCTCCOλT CβTTOTCλβλ λGTλλβTTββ CCβCλβTβTT 9000 λTCλCTCλTβ βTTλTGβCAG CλCTGCλTλλ TTCTCTTλCT GTCλTGCCAT CCGTλλGATG 9060 CTTTTCTβTβ λCTCGTβλGT λCTCλλCCλλ βTCλTTCTCλ GλATλGTGTλ TβCCβCβλCC 9120 βλGTTGCTCT TβCCCβGCCT CλλCACCGGλ TλλTλCCβCβ CCλCλTλβCλ GλλCTTTλλλ 9180 AGTGCTCλTC λTTGCλλλλC βTTCTTCGGG GCβλλλλCTC TCλλββλTCT TλCCGCTGTT 9240 βλβλTCCλCT TCGλTGTλλC CCλCTCGTGC λCCCλλCTβλ TCTTCAGCAT CTTTTλCTTT 9300 C CCAGCGTT TCTGGGTGλG CλλλλλCλββ λλββCλλλλT GCCβCλλλλλ λβββλλTλλβ 9360 GβCβλCλCGβ λλATGTTGAA TλCTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA 9420 TCAGGGTTλT TGTCTCλTGA GCGGATACAT λTTTGλλTGT ATTTAGAAAA λTλλλCλλλT 9480 AGGGGTTCCG CGCλCΛTTTC CCCβλλλλβT GCCACCTGAC GTCTλλGλλλ CCATTATTAT 9540 CATGACATTA λCCTλTλλλλ λTλGGCGTAT CλCβλββCCC TTTCGTCTTC λλ 9592 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 baae paire (B) TYPE: nucleic acid (C) STRλNDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TλβTλλλTTT GGGC 14 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 14 baee paire (B) TYPE: nucleic acid (C) STRλNDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AGTAAGATTT GGCC 14 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 14 baee paire (B) TYPE: nucleic acid (C) STRλNDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AβTβλλλTCT GλλT 14 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 14 baee paire (B) TYPE: nucleic acid (C) STRλNDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: βλλTλλTTTT GTGT 14 (2) INFORMATION FOR SEQ ID NC:8i (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 14 baee paira (B) TYPE: nucleic acid (C) STRλNDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGTAATλTTT GTCT 14

Claims: WHAT IS CLAIMED IS: 1. A recombinant viral vector comprising: (a) the DNA of, or corresponding to, at least a portion of the genome of an adenovirus, which portion is capable of infecting a hepatic cell; (b) a human VLDL receptor gene operatively linked to regulatory sequences directing its expression, said gene flanked by the DNA of (a) and capable of expression in the hepatic cell. 2. The vector according to claim 1 wherein said adenovirus DNA comprises the adenovirus 5' and 3' cis-elements necessary for replication and virion encapsidation in the absence of sequence encoding viral genes. 3. The vector according to claim 1 or 2 wherein said adenovirus genome has a deletion in all or a part of the El gene. 4. The vector according to any of claims l to 3 wherein said adenovirus genome has a deletion in all or a part of the E3 gene. 5. The vector according to any of claims l to 3 wherein said adenovirus genome comprising deletions in the DNA sequences of all or a portion of the adenovirus genes selected from the group consisting of the E2a gene, the E4 gene, the late genes LI through L5, the intermediate genes IX and IV,, and a combination thereof. 6. A mammalian hepatocyte which expresses a human VLDL receptor gene introduced therein through transduction of the vector of any one of claims 1 to 5. 7. Use of a recombinant viral vector for the manufacture of a medicament, said viral vector comprising: (a) the DNA of, or corresponding to, at least a portion of the genome of an adenovirus, which portion is capable of infecting a hepatic cell; (b) a human VLDL receptor gene operatively linked to regulatory sequences directing its expression, said gene flanked by the DNA of (a) and capable of expression in the hepatic cell. 8. The use according to claim 7 wherein the medicament is used for reducing cholesterol levels in familial hypercholesterolemia patients. 9. The use according to claim 7 wherein the medicament is used for reducing cholesterol levels in familial combined hyperlipide ia patients.

Inventors: KOZARSKY KAREN, STRAUSS JEROME III, WILSON JAMES M, KOZARSKY, KAREN, STRAUSS, JEROME, III, WILSON, JAMES, M.
Applicants: KOZARSKY KAREN, STRAUSS JEROME III, UNIV PENNSYLVANIA, WILSON JAMES M, KOZARSKY, KAREN, STRAUSS, JEROME, III, THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, WILSON, JAMES, M.
Application year: 1996
Main classification: C12N15
IPC codes: A61K35, A61K38, A61K48, A61P3, C07K14, C12N5, C12N7, C12N15