WO2001005943A2 - Compositions a base de lignees cellulaires de recombinaison secretant lcat, et methodes associees - Google Patents

Compositions a base de lignees cellulaires de recombinaison secretant lcat, et methodes associees Download PDF

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WO2001005943A2
WO2001005943A2 PCT/US2000/019047 US0019047W WO0105943A2 WO 2001005943 A2 WO2001005943 A2 WO 2001005943A2 US 0019047 W US0019047 W US 0019047W WO 0105943 A2 WO0105943 A2 WO 0105943A2
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cells
lcat
composition
cell
nucleic acid
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PCT/US2000/019047
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WO2001005943A3 (fr
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Anice E. Thigpen
Steven B. Lane
Thomas C. Becker
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Betagene, Inc.
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Publication of WO2001005943A3 publication Critical patent/WO2001005943A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates generally to the fields of cellular biochemistry and recombinant expression and to cholesterol metabolism. More particularly, the invention provides compositions, kits and devices comprising engineered cells that secrete therapeutically effective levels of lecithin cholesterol acyltransferase (LCAT). Methods of using such cells in various diagnostic and therapeutic embodiments are also provided, including the use of surprisingly effective low doses to treat LCAT deficiencies, such as those connected with atherosclerosis.
  • LCAT lecithin cholesterol acyltransferase
  • Lecithin cholesterol acyltransferase is a plasma protein enzyme that catalyzes the transfer of fatty acid from the sn-2 position of lecithin to the free hydroxyl group of cholesterol (Glomset et al, 1966).
  • the esterification process mediated by LCAT has long been presumed to be the key step in transferring cholesterol from the tissues of the body to the liver. This process, termed “reverse cholesterol transport", facilitates cholesterol removal from the body (Glomset, 1968).
  • Classic LCAT Deficiency (or Classic LCAT Deficiency Disease or Syndrome), which is characterized by clouding of the cornea, normochromic anemia and glomerulosclerosis. Mutations in the LCAT gene that result in some residual LCAT activity, including heterozygous LCAT mutations, lead to Fish-Eye Disease, typified by opacity of the cornea and hypoalphalipoproteinemia (Klein et al, 1992).
  • Atherosclerosis is a pathological condition characterized by excess cholesterol that accumulates in the arteries. In the atherosclerotic process, cholesterol accumulates in the foam cells of the arterial wall, thereby narrowing the vessel lumen and decreasing blood flow.
  • the clinical manifestations of atherosclerosis include coronary heart disease, heart attacks, strokes and peripheral vascular diseases, which have a very significant social and economic impact in the Western World.
  • LCAT high density lipoproteins
  • HDL high density lipoproteins
  • free cholesterol free cholesterol
  • the present invention addresses the foregoing and other drawbacks inherent in the prior art by providing engineered cells, kits and implantable devices for diagnostic, prophylactic and therapeutic applications in patients with atherosclerosis and other conditions associated with LCAT deficiency.
  • the invention is based, in part, on the provision of recombinant host cells that secrete biologically effective levels of lecithin cholesterol acyltransferase (LCAT) and that function effectively in vitro and in vivo and, in part, on the design of new therapeutically effective doses, orders of magnitude below any previously considerable possible.
  • LCAT lecithin cholesterol acyltransferase
  • the invention thus overcomes the lack of motivation to develop cell-based therapies for LCAT deficiencies by circumventing the difficulties previously believed to exist in the art, particularly by counteracting the former belief that very high levels of LCAT expression need to be obtained.
  • the invention also provides recombinant host cells that function surprisingly effectively in implantable devices and that secrete prophylactically and therapeutically effective levels of LCAT.
  • the recombinant cells of the invention also address the long felt need in the art to provide a unified diagnostic and therapeutic system for efficiently implementing LCAT therapy in patients with atherosclerosis, coronary heart disease, stroke and peripheral vascular disease, despite the multifactorial nature of these conditions.
  • the cells and kits of the invention are thus useful in the pre-selection of patients who would most benefit from cell-based LCAT therapy and in the optimization of the therapeutic doses needed.
  • the invention thus provides compositions, kits, devices and methods comprising recombinant cells that express therapeutically effective amounts of LCAT, particularly realistic therapeutically effective amounts of LCAT.
  • the "recombinant" or “engineered” cells of the invention are cells that comprise at least a first LCAT transgene and that express and secrete the
  • the LCAT-expressing cells of the invention most preferably produce and secrete biologically active LCAT.
  • biologically active LCAT is LCAT that is sufficiently biologically active to provide a therapeutic benefit upon provision of the LCAT-expressing cells to an animal in need of LCAT therapy.
  • maintenance of biological activity is not required to practice the invention.
  • the minimal biologically active LCAT in therapeutic terms is thus LCAT with an activity that is sufficient to provide a therapeutic benefit upon provision of the maximum possible number of LCAT-expressing cells to an animal. Where the number of recombinant cells required to provide a therapeutic benefit becomes prohibitively large, the LCAT has fallen below the required activity level for the therapeutic aspects of the invention.
  • LCAT glycosylation pattern that tends towards the LCAT glycosylation pattern observed in plasma will generally be preferred for use in the invention.
  • Recombinant LCAT with glycosyl units that approximate to those in naturally occurring LCAT will have a higher specific activity, approaching the specific activity of normal LCAT. Although variability in activity is well tolerated, as described above, fidelity of glycosylation is preferred for producing LCAT with high specific activity.
  • the recombinant or engineered cells of the invention grow effectively in culture in vitro.
  • such recombinant or engineered cells are amenable to genetic engineering, more preferably, to multiple rounds of genetic manipulations or "iterative engineering”.
  • An additional preferred property is that the cells of the invention exhibit a phenotype sufficiently stable to allow effective propagation and use, preferably repeated use.
  • the "stable" phenotype is most important in maintaining the ability to secrete LCAT, i.e., the cells stably secrete LCAT.
  • Exemplary cells are stable cells and immortalized cells.
  • the recombinant cells of the present invention are preferably implantable cells.
  • implantable cells are cells that function sufficiently well in implantable biomedical devices to enable the expression, secretion and provision of sufficiently therapeutically effective amounts of LCAT in vivo.
  • the recombinant or engineered cells of the invention thus preferably "survive and function upon implantation in vivo".
  • Device survival is exemplified by the ability of a cell to exhibit high density growth and low nutrient requirements, which are therefore preferred properties for the cells of the invention.
  • the cells of the invention function at effective, significant, or even at substantially full occupancy in an implantable device in vivo.
  • the cells of the invention have "in vivo longevity". Although included within selectively permeable devices, it is advantageous for the implanted cells to be resistant to small molecule mediators of immune damage. Yet a further preferred property of the cells of the invention is therefore being "cytokine resistant”.
  • Certain preferred engineered or recombinant cells of the invention are therefore those that comprise an LCAT transgene, grow effectively in culture, function effectively in implantable devices in vivo, have a sufficiently stable phenotype, stably secrete LCAT, are amenable to genetic engineering, and that express therapeutically effective amounts of biologically effective LCAT, particularly realistic therapeutically effective amounts of biologically effective LCAT.
  • the preferred LCAT-expressing cells are thus "culturable, implantable, sufficiently stable, stably secrete LCAT, engineerable and produce therapeutic LCAT levels".
  • compositions comprising engineered LCAT-expressing cells.
  • the compositions may be in vitro culture media compositions (including adherent cultures and suspension cultures), compositions suitable for freezing and thawing, compositions suitable for bulk LCAT production or pharmaceutically acceptable compositions.
  • the compositions may be aliquoted, packaged into kits, combined with other agents or therapeutics and/or comprised within implantable medical devices.
  • the in vitro culture of the cells of the invention may be conducted in defined media, such as that described and enabled in co-pending U.S. patent application Serial No. 09/228,503 and co-pending PCT patent application Serial No. PCT/US99/00633, each filed January 11, 1999 and specifically incorporated herein by reference.
  • the defined media may be further supplemented with serum and/or a growth factor that acts on the cell, such as fetal bovine serum, HGF, IGF-1, PDGF, NGF or growth hormone, as described in the foregoing applications.
  • compositions within a composition of the invention will be engineered cells expressing recombinant LCAT.
  • the compositions will thus comprise a plurality or a population of engineered or recombinant cells that express LCAT.
  • the engineered LCAT-expressing cells will form the majority of the cells in the composition.
  • the cell compositions may comprise native or engineered cells other than the LCAT- expressing cells of the present invention.
  • An exemplary aspect of the invention is a composition comprising a plurality or a population of engineered or recombinant cells that comprise at least a first exogenous nucleic acid segment that expresses LCAT, wherein:
  • the cells are ⁇ G H04 (ATCC NCI H1299/CRL-5803) cells; or
  • the cells express and secrete only the amount of LCAT necessary to generate a therapeutic response upon provision to an animal in need of LCAT therapy.
  • ⁇ G H04 cells are ⁇ G H04 (ATCC NCI H1299/CRL-5803) cells. ⁇ G H04 cells are obtained from ATCC (CRL-5803) lung carcinoma cells with secretory features.
  • LCAT- expressing ⁇ G H04 cells are ⁇ G H04 cells that comprise at least a first exogenous nucleic acid segment that encodes LCAT and that express and secrete any therapeutically effective amount of LCAT. The LCAT-expressing ⁇ G H04 cells of the invention are thus not limited to expressing "only the minimal" amount of LCAT necessary to generate a therapeutic response.
  • LCAT-expressing ⁇ G H04 cells are particularly advantageous as they grow well in culture, are phenotypically stable, stably secrete LCAT, amenable to multiple rounds of genetic engineering and survive and function well in implantable devices. These cells further possess the necessary post-translational processing capacity to produce biologically active recombinant human LCAT with a specific activity that approximates to that of LCAT in human plasma and that is believed to have a glycosylation pattern similar to the glycosylation pattern of native human LCAT.
  • Cells that express "only the amount of LCAT necessary to generate a therapeutic response upon provision to an animal in need of LCAT therapy” are generally cells that express “biologically sufficient, but reduced doses” or “biologically effective low doses” of LCAT. "Only the amount of LCAT necessary” means approximately only the amount necessary to generate a therapeutic response, without the unnecessary cost and practical burden of providing LCAT in excess of therapeutic needs.
  • the present invention therefore provides both the motivation and means to achieve successful therapy, not believed to be realistic on previous evidence, and provides efficient, labor-saving, cost-effective and widely applicable therapy.
  • LCAT or LCAT-expressing cells necessary to generate a therapeutic response are exemplified by amounts of LCAT or LCAT-expressing cells effective to increase the HDL/LDL ratio upon contact with a sample comprising HDL (high density lipoproteins) and LDL (low density lipoproteins).
  • the "HDL/LDL ratio” is precisely termed the HDL(mature)LDL ratio, which is increased upon contact with a sample comprising total HDL (including nascent HDL) and LDL.
  • the increase may be readily determined in vivo, wherein provision of the cells to, or implantation of the cells into, an animal or patient will bring the cells and the expressed LCAT into contact with the sera, which comprises HDL and LDL.
  • the capacity to increase HDL/LDL ratios in vivo can also be conveniently reproduced in vitro, upon contact of the cells with a suitable sample.
  • the recombinant LCAT-expressing cells of the invention, or cell supernatant or media conditioned by such cells, are thus tested in vitro for the capacity to mature HDL particles upon contact with a sample.
  • An ability to mature HDL particles in vitro is thus indicative of an ability to increase HDL/LDL ratios in vivo.
  • the low but effective doses of LCAT provided by the present invention are orders of magnitude below any previously contemplated in a hypothetical sense.
  • the normal LCAT activity in human sera is typically about lOO nmol/ml/hr, said to range from about 60 nmol/ml/hr to about 130 nmol/ml/hr.
  • the present invention contemplates that only about 5%, 10%), 20% up to only about 50% of the normal levels need to be supplied to provide a therapeutic benefit. Taking the 100 nmol/ml/hr value as normal, 5% to 50% is 5 nmol/ml/hr to 50 nmol/ml/hr.
  • 5% to 50% is 3 nmol/ml/hr to 30 nmol/ml/hr.
  • the present inventors consider 130 nmol/ml/hr as above normal in most circumstances, but note that 5%, 10%, 20%, 30% and 40% of 130 nmol/ml/hr is 6.5, 13, 26, 39 and 52 nmol/ml/hr, respectively.
  • compositions of the invention should comprise a population or plurality of cells that comprise at least a first exogenous nucleic acid segment that express sufficient LCAT to increase the serum LCAT activity to between about 1 nmol/ml/hr and about 50 to 55 nmol/ml/hr, to between 5 nmol/ml/hr and about 50 to 55 nmol/ml/hr, to between about 10 nmol/ml/hr and about 50 to 55 nmol/ml/hr, or to between about 20 nmol/ml/hr and about 50 to 55 nmol/ml/hr, upon administration of the cells to an animal, mammal or human.
  • Ranges of between about 1 and 30, 1 and 40, 1 and 50, 1 and 55, 3 and 30, 3 and 40, 3 and 50, 3 and 55, 6.5 and 30, 6.5 and 40, 6.5 and 50, 6.5 and 55, 13 and 30, 13 and 40, 13 and 50, 13 and 55, 26 and 40, 26 and 45, 26 and 50, 26 and 55 nmol/ml/hr are included.
  • compositions comprising cells that express recombinant LCAT in amounts sufficient to increase the serum LCAT activity to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56 or so nmol/ml/hr are included.
  • One "unit" (“U) of LCAT activity is defined as the amount of LCAT that converts one nmol of cholesterol to cholesterol ester per hour. Therefore, nmol/ml/hr are the same as U/ml.
  • the cells may express more than the minimal therapeutically effective amount of LCAT, i.e., more than "only the necessary" amount.
  • the ⁇ G H04 cells of the invention are not limited to producing minimally effective amounts of LCAT.
  • the preferred, culturable, implantable, sufficiently stable, stably secreting LCAT and engineerable cells of the invention may thus express recombinant LCAT in amounts sufficient to increase the serum LCAT activity to about 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or so nmol/ml/hr upon administration of the cells to an animal, mammal or human.
  • compositions comprising cells that express only the minimal therapeutic amount of LCAT may comprise ⁇ G H04 cells, but they need not.
  • the "cells other than ⁇ G H04 cells” may rather be of virtually any cell type that is engineerable and capable of secreting a product.
  • the cells are preferably engineerable, capable of secreting therapeutic LCAT levels, culturable, implantable, sufficiently stable and stably secrete LCAT.
  • the cells may be of hepatic origin or non-hepatic origin.
  • the present invention shows that cells of non-hepatic origin are capable of secreting LCAT with high specific activity, believed to result from normal or near-normal glycosylation.
  • This effective use of engineered cells of non-hepatic origin is surprising in that LCAT is naturally produced by hepatic (liver) cells.
  • Hepatic origin cells may also function well in certain aspects of the invention. Examples of hepatic cells are HepG2 cells, although larger numbers of engineered HepG2 cells will generally be needed to counteract the lower LCAT specific activity resulting from a glycosylation pattern different to plasma LCAT.
  • Cells for use with the invention should have a secretory capacity. These include cells with a constitutive secretory pathway and those with a regulated secretory pathway. Exemplary cells are secretory cells, such as neuroendocrine cells. Secretory and neuroendocrine cells are particularly preferred for use with co-expression embodiments.
  • a "secretory cell”, as used herein, is a cell that has a regulated secretory pathway.
  • the invention thus provides engineered secretory or neuroendocrine cells that grow effectively in culture, function well in implantable devices in vivo, have substantially stable phenotypes, are amenable to genetic engineering and stably express at least a first LCAT transgene at levels sufficient to secrete a therapeutically effective amount of the encoded LCAT enzyme when an implantable device comprising a population of such cells is provided to an animal, mammal or human.
  • Suitable secretory cells are neuroendocrine cells and endocrine cells, including pituitary cells, pancreatic beta cells and pancreatic alpha cells. Fetal cells and primary cells may be used if desired. Human cells are often preferred for human therapy. However, a wide range of non-human and mammalian cells, including rodent cells such as mouse, rat and hamster cells, may be employed, particularly when encapsulated in implantable devices. Human neuroendocrine cells and stable, human neuroendocrine cells are particularly preferred. Insulinoma cells, such as INS-1 cells, are particular examples.
  • the invention is not limited to the foregoing cells, and other useful cells include lung, connective tissue cells, gastrointestinal, pancreatic, pituitary, cecum, colon, thyroid, bladder, insulinoma, neuroectodermal and gastric cells.
  • Certain exemplary host cells are ⁇ TC, RIN, HIT and AtT20 cells (U.S. Patent No. 5,427,940, specifically incorporated herein by reference), PC12 (ATCC CRL 1721), NCI-H810 (CRL-5816), BON, NCI-H508 (CLL-253) and GH3 (ATCC CCL82.1) cells.
  • FIG. 251 For exemplary host cells, ⁇ G HOI (ATCC CCL-251), ⁇ G H02 (ATCC CRL- 1803), ⁇ G H03 (ATCC CRL-5816, NCI-H810), ⁇ G H04 (ATCC CRL-5803), ⁇ G H05 (ATCC CRL-5808), ⁇ G H06 (ATCC CRL-5815), ⁇ G H07 (ATCC CRL-5804), ⁇ G H08 (ATCC CRL- 5867), ⁇ G H09 (ATCC CRL-5838), ⁇ G H10 (ATCC CCL-185), ⁇ G Hl l (ATCC HTB-9), ⁇ G H12, ⁇ G H13 (ATCC CRL-2139), ⁇ G H14 (ATCC CCL-249), ⁇ G H15 (ATCC CCL-253), ⁇ G H16 (ATCC CRL-5974), ⁇ G H17 (ATCC CRL-5974), ⁇ G H18 (ATCC HTB
  • the cells of the invention are engineered or recombinant cells that comprise at least a first LCAT transgene or recombinant LCAT gene.
  • "Engineered” or “recombinant” cells are thus cells into which a "recombinant” or exogenous gene has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man.
  • an "LCAT transgene” or “recombinant LCAT gene” is an exogenous nucleic acid, DNA segment or coding region that encodes LCAT and that expresses the encoded LCAT when provided to a host cell under conditions suitable for recombinant expression.
  • LCAT genes or transgenes will generally, although not exclusively, be in the form of a cDNA gene, i.e., they will not contain introns or large stretches of genomic or chromosomal DNA.
  • exogenous identifies a nucleic acid as being added by the hand of man. "Exogenous” can thus mean that the nucleic acid is provided to a cell in which it is not normally expressed. However, “exogenous” also includes the provision of additional copies of a gene to a cell that normally expresses at least some of the same product from an "endogenous" gene, i.e., one naturally present in the cell.
  • Exogenous genes, transgenes and nucleic acids also include genes encoding proteins of other species, such as human genes expressed in rodent cells and such like. "Exogenous” copies further include transgenes and coding segments positioned adjacent to promoters not naturally associated with expression of the gene in the natural environment.
  • exogenous transgene is generally used to identify nucleic acids as “exogenous”, rather than being used to differentiate between genomic and cDNA sequences. Nonetheless, the use of cDNA sequences is preferable as it is generally more convenient to employ cDNA constructs that entire genes. However, given that the entire gene sequence is known (McLean et al., 1986b, specifically inco ⁇ orated herein by reference), the possibility of employing a genomic version of LCAT is by no means excluded.
  • compositions, cells, kits and devices of the invention may express LCAT from virtually any mammalian species, such as murine, bovine, porcine or the like, although the human sequence is preferred for use in human therapy.
  • LCAT nucleic acids from non-human species may be used and engineered to improve expression by using at least a first codon optimized for expression in human cells.
  • the expressed LCAT protein will exhibit LCAT activity and may include an amino acid sequence of at least about 70%, 75%, 80%, 85%, 90%, 95%, 99% or even 100% identity to the sequence of SEQ ID NO:2.
  • the expressed LCAT may also be an engineered human LCAT protein, for example, an LCAT variant in which Ser at position 216 of the mature amino acid sequence is replaced by Ala.
  • the LCAT nucleic acids within the cells will encode biologically active LCAT proteins and may include an LCAT nucleic acid sequence that hybridizes to the human LCAT sequence provided herein as SEQ ID NO:l and available in GenBank under Accession number Ml 2625.
  • LCAT nucleic acids that hybridize to the nucleic acid sequence of SEQ ID NO:l under relatively stringent conditions will be preferred.
  • the LCAT nucleic acids may also comprise a nucleotide sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 99% or even 100% identical to the sequence of SEQ ID NO:l.
  • the cells of the invention may also include an LCAT nucleic acid sequence as described by McLean et al., 1986a or U.S. Patent No. 5,048,488, each specifically incorporated herein by reference, or may include the GenBank cDNA sequence of Ml 2625. Nucleic acid sequences encoding the so-called human LCAT-like protein of PCT patent application WO 98/46767 may also be employed.
  • LCAT is preferably, although not exclusively, provided to the cell in the form of an expression vector.
  • the exogenous LCAT nucleic acid segment may be stably integrated into the genome of the host cell(s).
  • LCAT expression from "endogenous" promoters is included, such as that which drives high LCAT expression after a "promoter trap" transfection approach.
  • the exogenous LCAT nucleic acid is operatively linked to at least a first exogenous promoter that expresses the LCAT nucleic acid in the host cell.
  • the engineered cells of the invention may thus comprise at least a first exogenous nucleic acid construct that comprises at least a first nucleic acid sequence that encodes LCAT operatively positioned downstream from at least a first exogenous promoter that directs expression of the encoded LCAT.
  • exogenous promoters include constitutive promoters, inducible promoters and tissue-specific and tissue-preferential promoters, such as neuroendocrine cell- specific and neuroendocrine cell-preferential promoters.
  • Constitutive promoters may be preferred in certain embodiments in order to direct high-level expression.
  • Exemplary promoters include CMV, SV40 IE, RSV LTR, GAPDH, ubiquitin, MMLV-LTR, RIP1, multimerized RIP and HIP promoters, of which the CMV promoter is currently preferred.
  • the exogenous LCAT nucleic acids and transgenes may be provided to the cells in the form of expression constructs such as a plasmid, cosmid or viral expression vector.
  • the expression constructs and vectors may be incorporated into the cell by electroporation, calcium precipitation or by formulation in a liposome and cellular uptake of the liposome.
  • Exemplary viral expression vectors for providing the exogenous nucleic acids by viral infection include adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, hepatitis viral vectors, retroviral vectors, lentiviral vectors and herpes viral vectors.
  • the expression plasmids or vectors will preferably comprise the control elements necessary for efficient expression, such as polyadenylation signals and the like.
  • the cells may also contain and express an LCAT transgene in place of an endogenous gene, the expression of which endogenous gene has been inhibited, mostly by "gene knockout".
  • the cells may include an LCAT transgene in the form of knockout and replacement technology.
  • Candidate genes for knockout may include genes for other highly expressed and/or secreted products and/or genes that express products that are undesirable for therapy and/or human administration.
  • the cells of the invention may further comprise additional exogenous or recombinant genes.
  • additional exogenous or recombinant genes In fact, other than the practical limitations of genetic engineering, there is virtually no limit to the number of exogenous genes that may be included.
  • the cells may thus comprise at least a second, third, fourth, fifth, sixth or so, exogenous nucleic acid segment that expresses a second, third, fourth, fifth, sixth or so product, generally a translated product such as a protein, polypeptide or peptide.
  • the first (LCAT) and second exogenous nucleic acid segments may each be comprised on the same expression construct, or they may be provided to the same cell on distinct expression constructs.
  • An IRES may be located between the LCAT coding region and the at least a second coding region.
  • the at least a second coding region may be operatively linked to a second exogenous promoter.
  • the additional exogenous nucleic acid segments may expresses a therapeutic protein, polypeptide or peptide.
  • exemplary therapeutic products include secreted products, such as amidated polypeptides, glycosylated polypeptides, other secreted enzymes, hormones, cytokines, growth factors, cholesterol lowering agents and the like.
  • Other therapeutic products include those that are not necessarily secreted, but that impart a useful property to the host cell. These include agents to facilitate immune resistance and cell surface receptors that increase the sensitivity of the cell to pharmacological or physiological modulators of secretion, thereby providing an additional control mechanism for LCAT secretion.
  • hormones for combined use with the invention include growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, ⁇ -endorphin, ⁇ -melanocyte stimulating hormone ( ⁇ -MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, insulin, amylin, lipotropins, GLP- 1 , neurophysins and somatostatin.
  • growth hormone prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin (ACTH), angiotensin I, angiotensin
  • Insulin, growth hormone, glucagon, amylin and GLP-1 are preferred examples. Where insulin secretion is concerned, human insulin is preferred and the cell should ideally secrete insulin in response to glucose.
  • the ability to secrete insulin in response to glucose may be "re-engineered” by providing one or more of recombinant insulin, GLUT-2 and/or glucokinase genes, as described in U.S. Patent No. 5,427,940, specifically incorporated herein by reference.
  • Such cells may also have reduced hexokinase I activity relative to the cells from which they are prepared.
  • Suitable amidated polypeptides for combined use include calcitonin, calcitonin gene related peptide (CGRP), ⁇ calcitonin gene related peptide, hypercalcemia of malignancy factor (1-40) (PTH-rP), parathyroid hormone-related protein (107-139) (PTH-rP), parathyroid hormone-related protein (107-111) (PTH-rP), cholecystokinin (27-33) (CCK), galanin message associated peptide, preprogalanin (65-105), gastrin I, gastrin releasing peptide, glucagon-like peptide (7-36 amide) (GLP-1 (7-36 amide)), pancreastatin, pancreatic polypeptide, peptide YY, PHM, secretin, vasoactive intestinal peptide (VIP), oxytocin, vasopressin (AVP), vasotocin, enkephalins, enkephalinamide, metorphinamide (a
  • Exemplary growth factors for combination with the invention include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial cell growth factor (VEGF), transforming growth factor- ⁇ (TGF- ⁇ ), hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1).
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • VEGF vascular endothelial cell growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • HGF hepatocyte growth factor
  • IGF-1 insulin-like growth factor 1
  • the second transgene may encode adenosine deaminase, galactosidase, glucosidase, factor IX, sphingolipase, lysosomal acid lipase, lipoprotein lipase, hepatic lipase, pancreatic lipase related protein, pancreatic lipase or uronidase.
  • the present invention is ideally suited to iterative engineering of LCAT itself, as well as LCAT in combination with other agents.
  • the at least a second, third, etc. exogenous coding region may therefore also express LCAT in addition to the first exogenous coding region.
  • Cholesterol lowering agents may be co-expressed in combination with the invention. These include agents that up-regulate the endogenous production of LCAT and components such as apolipoprotein A-I (Apo A-I), which decrease the accumulation of cholesterol.
  • Apo A-I apolipoprotein A-I
  • cell surface receptors in the LCAT-expressing cells of the invention are particularly contemplated. Suitable examples include ⁇ -adrenergic receptor, ⁇ - adrenergic receptor, sulphonylurea receptor (SUR) or inward rectifying potassium channel (Kir) subunit of the KATP channel, GLP-1 receptor, growth hormone receptor, arginine vasopressin receptor, luteinizing hormone receptor, corticotropin receptor, urocortin receptor, pancreatic polypeptide receptor, glucocorticoid receptor, somatostatin receptor, muscarinic receptor, calcium channel, voltage-gated channel, BK channel and leptin receptor.
  • SUR sulphonylurea receptor
  • Kir inward rectifying potassium channel subunit of the KATP channel
  • GLP-1 receptor growth hormone receptor
  • arginine vasopressin receptor luteinizing hormone receptor
  • corticotropin receptor urocortin receptor
  • pancreatic polypeptide receptor glucocor
  • the selectable marker gene may be flanked by LoxP sites.
  • the additional coding region(s) will encode at least a first "suicide gene” or “kill system”, otherwise known as “negatively selectable marker gene(s)". At least a first negatively selectable marker gene is provided for targeted cell killing.
  • the objective of the suicide genes, systems and negatively selectable markers for targeted cell killing is to provide a safety net for cellular therapeutic applications in which non-functioning cells and/or escaped cells may be specifically and safely destroyed.
  • Exemplary negatively selectable marker genes include cytosine deaminase, HSV-thymidine kinase, GLUT-2 and nitroreductase.
  • an IRES may be located between the second coding region and third coding region.
  • An IRES may be located between the LCAT coding region, the second coding region and the third coding region.
  • the third coding region may be operatively linked to a third exogenous promoter.
  • the third exogenous coding regions may comprise any one or more of the foregoing therapeutic proteins, polypeptides, peptides or agents, such as amidated polypeptides, glycosylated polypeptides, secreted enzymes, hormones, cytokines, growth factors, cholesterol lowering agents, immune resistance components and cell surface receptors. Combinations of one additional therapeutic product and one suicide gene or negatively selectable marker are preferred.
  • an additional preferred feature of the invention is the use of two and three suicide genes, integrated independently using independent promoters.
  • Certain preferred aspects of the invention concern cells comprising a combination of at least two exogenous LCAT nucleic acid segments and at least one exogenous nucleic acid segment that express a negative selectable marker protein for targeted cell killing.
  • Particularly preferred are cells that comprise at least two exogenous LCAT nucleic acid segments and at least two negative selectable marker genes for targeted cell killing.
  • compositions of the invention may comprise at least a second, third, fourth, fifth, sixth, etc., therapeutic agent in a non-cell-based sense.
  • therapeutics include other agents that activate existing LCAT, remove inhibitors of LCAT, up-regulate the endogenous production of LCAT, and the like.
  • Further components are those that generally lower cholesterol levels, such as Apo Al and Apo AIL
  • Still further examples are any agents used in the treatment of any disorder directly or indirectly associated with LCAT deficiency, including medicaments for treating atherosclerosis and coronary heart disease.
  • U.S. Patents Nos. 5,736,157 and 5,746,223 are exemplary of such methods.
  • the cells and compositions of the invention are not limited to in vivo uses and may, e.g., be employed to produce LCAT in vitro.
  • Appropriate methods comprise culturing a population of recombinant LCAT-expressing cells in accordance with the present invention and obtaining the LCAT produced thereby.
  • the use of LCAT- expressing ⁇ G H04 cells is generally preferred as such cells are not designed to produce only the minimal amounts of LCAT required for efficient and effective therapy. Higher levels of LCAT can thus be obtained from cultures of ⁇ G H04 cells that express recombinant LCAT.
  • the LCAT-expressing cells may also be used in biological assays for the identification of novel therapeutic compounds and/or drug targets, i.e., as direct or indirect screening tools for the identification of secretory modulators for therapeutic use.
  • Co-pending U.S. patent application Serial No. 09/228,499 and co-pending PCT patent application Serial No. PCT/US99/00551, each filed January 11, 1999, are specifically inco ⁇ orated herein by reference for pu ⁇ oses of further describing and enabling such drug screening technology.
  • the drug screening technology may be conducted when the cells are located within an animal, as described in the foregoing applications.
  • the present invention concerns the generation of the recombinant LCAT-expressing cells in vivo or in situ.
  • the exogenous LCAT nucleic acid or gene is provided to the target cells simply by administering a suitable LCAT genetic construct to an animal or human in a manner effective to allow uptake and expression of the genetic construct.
  • Viral vectors and recombinant viruses are generally preferred for such embodiments, although techniques such as naked DNA delivery, biolistics and the like may be employed. Many viral vectors readily achieve expression in the liver, which is the normal site of LCAT expression, which is thus an advantage of the invention.
  • compositions and cells are engineered ex vivo and administered to an animal or human in a form that already comprises the exogenous LCAT gene.
  • the compositions and cells of the invention may thus be formulated in pharmaceutically acceptable diluents or vehicles and may also be preferably aliquoted or pre- prepared for inco ⁇ oration into kits and/or implantable medical or veterinary devices.
  • the cells may be encapsulated or microencapsulated in a biocompatible membrane, coating or matrix; fiber seeded into a semi-permeable fiber; or comprised within a biocompatible, implantable device.
  • the present invention thus further provides a range of biocompatible, implantable devices that comprise a population of cells, wherein at least a portion or sub-population of the cells are engineered, LCAT-expressing cells in accordance herewith.
  • the devices generally contain a number of cells sufficient to produce a therapeutic benefit upon implantation of the entire device, or a number of such devices, as appropriate, into an animal, mammal or human.
  • the type of device for use with the invention is virtually unlimited, so long as the device is sufficiently biocompatible to prevent untoward reactions upon provision to an animal or human and allows the release of the secreted LCAT.
  • the devices will most preferably be semi- permeable or selectively permeable, such that the secreted LCAT is provided to the animal, but that significant host components do not infiltrate the device.
  • Exemplary encapsulation and microencapsulation techniques include hydrogel and alginate coatings and fiber seeding into semi-permeable fibers.
  • the devices further include those where the cell population is positioned in a semi-permeable membrane, such as a tubular membrane, positioned within a protective housing.
  • a semi-permeable membrane such as a tubular membrane
  • Each end of the tubular membrane may be attached to an arterial graft that extends beyond the housing and joins the device to a vascular system as an arteriovenous shunt.
  • rods are currently preferred over microspheres.
  • the preferred flexible rods may be used in a linear manner or coiled to any desired format, including patches.
  • the invention further provides pharmaceutical and cell-based therapy dosage forms of LCAT-expressing recombinant cells.
  • the pharmaceutical dosage forms comprise a pharmaceutically acceptable carrier and a pharmacologically effective amount of LCAT- expressing recombinant cells in accordance with the invention.
  • the cell-based therapy dosage forms are preferred, and comprise a biocompatible matrix or implantable device and a pharmacologically effective amount of LCAT-expressing recombinant cells in accordance herewith.
  • the cell-based therapy dosage forms may comprise recombinant cells in accordance with the invention that secrete any amount of LCAT sufficient to provide the desired serum levels of LCAT activity. As taught herein, only about 5%, 10%, 20%) up to only about 50% of the normal levels need to be supplied in order to provide a therapeutic benefit.
  • the normal protein level of LCAT in human sera is typically about 5 ⁇ g/ml.
  • the "amounts" required to give the desired "activity" will depend on the specific activity of the LCAT produced by the recombinant cells, i.e., on the units of activity per mass of protein. However, as the present invention is able to prepare recombinant LCAT with a specific activity that approaches and/or corresponds to the specific activity of LCAT in human sera, a direct comparison on the mass level is appropriate.
  • the cell-based therapy dosage forms of the present invention need supply LCAT with an approximately normal specific activity in preferred amounts of only about 0.25 ⁇ g/ml, about 0.5 ⁇ g/ml or about 1.0 ⁇ g/ml, up to only about 2.5 ⁇ g/ml to provide a therapeutic benefit.
  • the cell- based therapy dosage forms may express sufficient LCAT to increase the serum LCAT levels to about 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or so ⁇ g/ml upon administration to a mammal. Levels of between about 0.25 and 0.5, 0.25 and 0.75, 0.25 and 1, 0.25 and 2, 0.5 and 0.75, 0.5 and 1, 0.5 and 2, 1 and 1.5, 1 and 1.75, 1 and 2 ⁇ g/ml are included.
  • compositions, cells, kits and devices of the invention may be employed in a variety of methods and uses in which LCAT is provided to an animal, generally a mammal or human, to increase serum LCAT levels.
  • the invention thus provides for the use of the compositions and cells in accordance herewith in the manufacture of medicaments for use in providing LCAT to a mammal or human, to increase serum LCAT levels.
  • the methods, uses and medicaments typically provide LCAT to mammals or humans "in need thereof.
  • the "need for LCAT” includes both therapeutic and prophylactic needs, such that the invention has uses in both treating and preventing conditions associated with LCAT deficiency.
  • Treatment is effected by providing a therapeutically effective amount of LCAT- expressing cells of the invention to a mammal or human having a condition associated with LCAT deficiency.
  • Preventative are implemented by providing a prophylactically effective amount of LCAT-expressing cells of the invention to a mammal or human at risk for developing a condition associated with LCAT deficiency.
  • Conditions associated with LCAT deficiency include Fish-Eye Disease or Syndrome and Classic LCAT Deficiency (or Classic LCAT Deficiency Disease or Classic LCAT Deficiency Syndrome), each of which may be treated by the present invention.
  • Further "conditions associated with LCAT deficiency” include prevalent clinical conditions, such as atherosclerosis, ischemic and coronary heart disease and heart attack, stroke and peripheral vascular disease, and any as yet-unknown conditions that prove to correlate with LCAT deficiency.
  • the methods, uses and application of medicaments are designed to provide to the animals and humans populations of LCAT-expressing cells in amounts to impart serum LCAT levels that are effective to treat or prevent the condition at issue.
  • Serum LCAT levels that are effective to treat or prevent conditions of LCAT deficiency may be exemplified by levels effective to decrease the accumulation of cholesterol in animals and humans, wherein the accumulation of cholesterol is an indicator of the condition. "Decreasing" the accumulation of cholesterol includes slowing and/or reversing the process of cholesterol accumulation in animals and humans, including the accumulation of cholesterol particularly in the arteries of the animals and humans. “Decreased” cholesterol is thus decreased accumulation of cholesterol in peripheral tissues and/or decreased LDL.
  • one aspect of the invention is the discovery of cells with a preferred combination of properties for in vivo use.
  • Such cells comprise at least one LCAT transgene and are culturable, implantable, sufficiently stable, engineerable and stably produce therapeutic LCAT levels, as exemplified by LCAT-expressing ⁇ G H04 cells.
  • the cells may be provided to the animals and humans in amounts to impart any therapeutically effective serum LCAT level and are not limited to the su ⁇ risingly low minimal therapeutic amounts disclosed herein.
  • the cells and medicaments will preferably be provided in an amount to impart only the
  • LCAT necessary to generate a therapeutic response, without wasteful excess.
  • exemplary therapeutically effective amounts of LCAT-expressing cells are those effective to increase the serum HDL/LDL ratio upon provision of the cells to, or implantation of the cells within, an animal or patient, thereby treating the underlying condition.
  • the present invention provides for an added level of control over LCAT secretion.
  • a cell that naturally or recombinantly expresses a cell-surface receptor that provides or increases the sensitivity of the cell to a modulator of secretion allows LCAT secretion to be controlled by variations in the level of the modulator.
  • Co-pending U.S. patent application Serial No. 09/228,500 and co-pending PCT patent application Serial No. PCT/US99/00631 are specifically inco ⁇ orated herein by reference for pu ⁇ oses of further describing and enabling the modulation of secretion in this manner.
  • receptor and modulator combinations that increase LCAT secretion will be employed, although the controlled decrease of LCAT secretion is by no means excluded.
  • Receptor and modulator combinations may be chosen so that the modulator is a naturally occurring (physiological) modulator that will increase LCAT secretion under certain physiological conditions. More preferably, receptor and modulator combinations are chosen so that the modulator is an exogenous (pharmacological) modulator that will increase LCAT secretion when the modulator is administered to the animal or human.
  • certain aspects of the invention concern methods, uses and medicaments for the controlled provision of LCAT to a mammal or human. These comprise providing to the mammal or human an LCAT-expressing cell that comprises a native or recombinant receptor that provides or increases the sensitivity of the cell to a secretory modulator, followed by providing the modulator to the mammal or human, thereby controlling LCAT secretion from the cell.
  • Appropriate receptors are SUR, Kir and SUR and Kir in combination, which may be used with modulators such as sulfonylureas and/or PrandinTM.
  • the range of methods within the invention may be combined with the use of one or more additional therapeutic agents, particularly those that increases LCAT levels or activity or that decrease cholesterol accumulation or otherwise combat atherosclerosis, heart disease and/or stroke.
  • All of the methods, uses and medicaments of the invention are particularly applicable to treating or preventing atherosclerotic conditions, wherein one may wish to conduct a "pre- screen" to identify an animal or human having or at risk for developing atherosclerosis, ischemic and coronary heart disease, heart attack, stroke and peripheral vascular disease.
  • the recombinant cells of the invention should then be provided or implanted in a manner effective to secrete an amount of LCAT effective to decrease cholesterol accumulation in the animal or human, thereby treating atherosclerosis, ischemic and coronary heart disease, heart attack, stroke and peripheral vascular disease.
  • Still further preferred aspects of the present invention concern the provision of methods and kits for use in diagnostic and prognostic investigations and in the design of therapeutic protocols.
  • the methods and kits may comprise the recombinant LCAT-expressing cells of the invention and/or cell supernatant or media conditioned by such cells; and are intended for use in testing the capacity of biological samples to mature the HDL particles upon contact with the cells or cell supernatant or conditioned media. It will be understood that an ability to "mature HDL particles in vitro" is indicative of an ability to increase HDL/LDL ratios in vivo.
  • kits are particularly important in connection with atherosclerosis, coronary heart disease and stroke due to the multifactorial nature of these conditions. Therefore, not all patients will be best treated by any single form of therapy, and the application of these aspects of the invention will allow appropriate groups of patients to be identified as most suitable for cell-based therapy. In addition, in identifying a patient suitable for treatment with recombinant LCAT-expressing cells, the in vitro tests provide valuable information as to the numbers of cells to be administered to produce a therapeutic benefit.
  • the invention provides methods for designing a therapeutic regimen for treating atherosclerosis, ischemic and coronary heart disease, heart attack, stroke and peripheral vascular disease, comprising testing a biological sample from a patient having or suspected of having any one or more of such conditions for the capacity to increase the HDL/LDL ratio upon contact with or exposure to engineered, LCAT-expressing cells in accordance with the invention.
  • LCAT-expressing cells in accordance with the invention, it will be understood that although LDL will decrease and HDL will increase upon contact with the LCAT-expressing cells, LDL is not "converted" to HDL, such that one does not become the other.
  • the capacity of the biological sample to allow a sufficient increase in the HDL/LDL ratio upon contact with the engineered cells or cell supernatant is indicative of a patient suitable for treatment with a population of the engineered cells.
  • the amounts of LDL and HDL in the sample are quantified; wherein a significant increase in the HDL/LDL ratio is indicative of a patient suitable for treatment with a population of the engineered cells and wherein a less significant increase in the HDL/LDL ratio is indicative of a patient that would be better treatment with a distinct anti-atherosclerotic therapy.
  • the biological samples are preferably serum samples.
  • kits comprising the recombinant LCAT- expressing cells of the invention.
  • the kits may comprise the cells in at least a first container.
  • the kits may alternatively or additionally comprise the cells and written instructions for use.
  • the kits may be therapeutic kits, wherein the kits may comprise a device or apparatus for administering a population of cells to a mammal or human.
  • Therapeutic kits may alternatively or additionally comprise the cells of the invention and at least a second therapeutic agent, generally an agent that stimulates LCAT activity or expression or that lower cholesterol levels by other mechanisms.
  • kits of the invention may be diagnostic kits, as described above.
  • the kits may comprise at least a first assay reagent for determining the amount of LDL or HDL, or both; and/or quantitated sample(s) of LDL or HDL, or both, to serve as a control.
  • These kits may again comprise written instructions for use.
  • FIG. 1 Effectiveness of H04 cells in implantation devices.
  • Cell lines expressing recombinant rat growth hormone were derived from human H03 cells (792/20) (closed squares), human H04 cells (782/17 R8) (closed circles) and RIN 1046-38 cells (660/49) (closed diamonds).
  • Cells were expanded in tissue culture, released by trypsinization and loaded into implantation devices. Devices were cultured under standard tissue culture conditions for up to 40 days. Media samples collected and assayed for rat growth hormone concentration. The gaps between data points for the RIN cells (closed diamonds) indicate loss of sample, not discontinuity of study.
  • statins which are HMG CoA reductase inhibitors.
  • LovastatinTM PravastatinTM and SimvastatinTM are the poor abso ⁇ tion, only 5%, 17% and 25% of these drugs reach the circulation, respectively, with the rest being excreted.
  • Bile acid sequestrants can also be used to lower LDL, but these drugs raise triglycerides.
  • CholestyramineTM (JAMA, 1984a) and ColestipolTM (JAMA, 1984b) can be used, although half of the patients treated refuse to continue treatment of either drug (JAMA, 1984a; 1984b).
  • Niacin probably has the best potential to raise HDL levels without raising triglycerides (JAMA, 1975). It has been reported to reduce LDL levels by about 20-30% and to elevate HDL by about 35-55%. The side effects are serious enough to be taken under physician's care though, and include rashes, ulcer irritation, gout, worsening of diabetes control and liver problems.
  • Other drugs that moderately lower LDL levels include GemfibrizolTM, ProbucolTM and ClofibrateTM (JAMA, 1975).
  • the present invention provides such compositions and methods in the form of implantable, recombinant cells that express LCAT and act to raise LCAT activity in the serum of patients.
  • the invention can thus also be employed to treat other conditions associated with LCAT deficiency, particularly Fish-Eye Disease and Classic LCAT Deficiency.
  • the invention can function to provide or maintain a ratio of total serum cholesterol to serum high density lipoproteins in a mammal at or below the level of average risk for heart disease, i.e., below five:one (Castelli et al, 1986).
  • the invention thus provides prophylactic and therapeutic treatment methods for use in combating atherosclerosis in patients by increasing the activity of LCAT in the serum of the patient to a rate effective to decrease the accumulation of cholesterol in the patients, in particular, to decrease the accumulation of cholesterol in the arteries of the subject.
  • the methods of the invention for treating other conditions involving LCAT deficiencies, such as Classic LCAT Deficiency and Fish-Eye Disease the methods also involve increasing the LCAT activity in the serum to a level sufficient to ameliorate the condition.
  • LCAT lecithin cholesterol acyltransferase
  • LCAT refers to an enzyme that catalyzes the synthesis of cholesterol esters and lysolecithin from phosphatidylcholine and unesterified cholesterol present in plasma lipoproteins. The enzyme is produced primarily by the liver and is found naturally in the serum of mammals and humans. LCAT is a "glycoprotein", which means that various carbohydrate units are attached at certain points of the polypeptide. Glycosylation is high in sialic acid and typically tribranched in LCAT in human plasma.
  • LCAT LCAT binds to antibodies
  • the mass of LCAT can be determined by ELISA or radioimmunoassay, such as a competitive double antibody radioimmunoassay.
  • Routine methods also are known for measuring absolute LCAT activity in biological samples and for measuring the more informative cholesterol esterification rate (see, e.g., Albers et al., 1986 and Gillett and Owens; each inco ⁇ orated herein by reference).
  • LCAT activity can be determined by measuring the conversion of radiolabeled cholesterol to cholesteryl ester after incubation of the enzyme and radiolabeled lecithin- cholesterol liposome substrates containing Apo A-I.
  • Endogenous cholesterol esterification rate can be determined by measuring the rate of conversion of labeled cholesterol to cholesteryl ester after incubation of fresh plasma that is labeled with a trace amount of radioactive cholesterol by equilibration with a [ 14 C] cholesterolalbumin mixture at 4°C.
  • the biological assay for LCAT function recreates the natural reaction (conversion of cholesterol to cholesterol ester on HDL particles) on artificial HDL particles ex vivo. These particles include cholesterol and a trace amount of labelled cholesterol, preferably radiolabelled cholesterol. After allowing an LCAT sample to react with the particles, the cholesterol and cholesterol esters are separated, e.g., by TLC, and measured, preferably radiometrically. The basic result is the percentage of the cholesterol that converted to cholesterol ester during one hour of the incubation. Knowing the amount of cholesterol in the reaction (e.g., 8 nmol), the percent conversion number can be used to calculate the nmol of cholesterol converted to cholesterol ester during a one hour period. This is the official definition of an LCAT unit (U), the nanomoles of cholesterol converted to cholesterol ester in 1 hour (nmol/hr).
  • the endogenous cholesterol esterification rate is a better measure of the therapeutic LCAT activity in vivo because it reflects not only the amount of LCAT activity present in the serum, but also the nature and amount of substrate and cofactors in the plasma. Thus, the cholesterol esterification rate is not necessarily proportional to either mass of LCAT or absolute
  • the standard LCAT activity in the sera of normal human subjects is generally accepted to be about 100 units/ml, with a range of 63-113 units/ml being reported.
  • Those of ordinary skill in the art understand that particular values should be inte ⁇ reted in light of the assay technique used. Although different assay techniques can produce varying results, these may be readily accounted for by the skilled artisan.
  • the present invention concerns the expression of isolated nucleic acids, DNA segments and genes encoding LCAT in recombinant host cells through the application of genetic engineering and recombinant DNA technology.
  • the LCAT nucleic acids, DNA segments and genes for use in the invention are isolatable from mammalian species, including humans.
  • the LCAT nucleic acids will be isolated free from total genomic DNA and capable of expressing a protein with LCAT activity upon provision to a recombinant host cell.
  • the LCAT nucleic acids and DNA segments will thus comprise LCAT coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences.
  • isolated substantially away from other coding sequences means that the LCAT nucleic acids and genes form the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • the term "gene” is used for simplicity to refer to a functional LCAT protein or polypeptide encoding unit.
  • this functional term includes both genomic sequences, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express, LCAT proteins and polypeptides.
  • Also included within the LCAT nucleic acids and DNA segments are recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Human LCAT cDNAs and genes have been cloned and sequenced (U.S. Patent No.
  • LCAT encoding and/or expressing LCAT
  • encoding and/or expressing LCAT refer to nucleic acid sequences that, upon transcription and translation of mRNA, produce proteins or polypeptides having LCAT enzyme activity and generally comprising amino acid sequences of LCAT.
  • cDNA, genomic DNA with introns removed upon transcription and processing into mRNA, and related degenerate sequences all "encode LCAT".
  • DNA sequences encoding LCAT can be obtained by any method known in the art.
  • coding sequences can be prepared by chemical synthesis.
  • PCRTM primers can be devised using the sequences of LCAT provided herein and LCAT cDNA or genomic DNA can be isolated by amplification.
  • GenBank cDNA sequence of the human LCAT gene (Ml 2625) is provided herein as SEQ ID NO:l.
  • the encoded protein sequence of human LCAT is also provided herein as SEQ ID NO:2.
  • the encoded LCAT protein has a leader sequence of 24 amino acids, which are cleaved to yield the mature product. Therefore, a sequence of 440 is the full length amino acid sequence and a sequence of 416 amino acids represents the cleaved product.
  • a number of particular expression constructs have been prepared and are described herein (Example 2).
  • Human LCAT constructs are preferred for use in the present invention, particularly in terms of human therapy. However, LCAT from other species may certainly be employed.
  • GenBank accession numbers for various species of LCAT that have been cloned and the percent homology of their protein sequence to the human sequence are shown in Table 3.
  • Human nucleic acid sequences encoding the LCAT-like protein of PCT patent application WO 98/46767 may also be employed if desired.
  • the LCAT genes for use in the invention may thus encode an LCAT protein that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:2, wherein the LCAT retains biological activity.
  • a "sequence essentially as set forth in SEQ ID NO:2” means that the sequence substantially corresponds to a portion of SEQ ID NO:2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2.
  • biologically functional equivalent is well understood in the art and is further defined in detail herein (see Table 4).
  • sequences that have between about 85% and about 90%, or more preferably, between about 91% and about 99%, of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO:2 will be sequences that are "essentially as set forth in SEQ ID NO:2". This is shown to be relevant in the present context as the murine and human sequences share 85% homology and clearly encode an LCAT protein with biological activity.
  • the LCAT genes for use in the invention may include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:l.
  • the term "essentially as set forth in SEQ ID NO:l” is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:l and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO:l.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (Table 4). Table 4
  • amino acid and nucleic acid sequences may include additional residues, such as additional N-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • sequences that have between about 85% and about 90%, or more preferably, between about 91 ) and about 99% of nucleotides that are identical to the nucleotides of SEQ ID NO:l will be sequences that are "essentially as set forth in SEQ ID NO:l". Sequences that are essentially the same as those set forth in SEQ ID NO:l may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:l under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art.
  • the present invention may utilize any mammalian LCAT or enzymatically active species or allelic variation thereof, including fragments of the enzyme that possess enzymatic activity.
  • a "species variant” of an LCAT protein, nucleic acid, DNA segment or gene is one in which the variation is naturally occurring among different species (Table 3).
  • An "allelic variant” of an LCAT protein, nucleic acid, DNA segment or gene is one in which alternative forms (alleles) exist within a given population, such as the human population.
  • allelic variants generally differ from one another by only one, or at most, a few amino acid substitutions. In the context of the present invention, it is important to use only "normal" allelic variants and not to use allelic variants associated with disease.
  • Biologically functional equivalent proteins are thus defined herein as those proteins in which certain, not most or all, of the amino acids may be substituted. Of course, a plurality of distinct LCAT proteins with different substitutions may easily be made and used in accordance with the invention.
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.
  • arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
  • hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8); tryptophan (0.9); tyrosine (1.3); proline (1.6); histidine (3.2); glutamate (3.5); glutamine (3.5); aspartate (3.5); asparagine (3.5); lysine (3.9); and arginine (4.5).
  • LCAT mutants engineered by the hand of man may therefore be employed in the invention, whether designed to be equivalent or not.
  • Francone et al. (1991; inco ⁇ orated herein by reference) described an LCAT variant in which Ser at position 216 of the mature amino acid sequence is replaced by Ala. This co ⁇ esponds to position 240 in SEQ ID NO:2 herein.
  • These and all related and other active mutants may be used in the present invention.
  • the present invention includes two unified features within the single inventive concept of effective cell-based therapy for LCAT deficiencies.
  • First, recombinant host cells are provided that have a combination of properties that make them su ⁇ risingly effective in therapeutic terms.
  • Second, a range of therapeutically effective doses are provided that are, unexpectedly, orders of magnitude below the previously contemplated hypothetical levels. In terms of the prefe ⁇ ed cell types, their use at any dosage level is appropriate. In terms of the low, but effective doses, the use of virtually any cell type is appropriate.
  • the two inventive themes of the present application are thus by no means mutually exclusive and their relative advantages allow for the effective treatment of the spectrum of LCAT-associated disorders.
  • Human cells are generally prefe ⁇ ed over other cells, such as rodent cells, for use in human therapy as cells transplanted within a species (allograft) are generally less susceptible to immunological destruction than cells transplanted across species (xenograft). Even in the context of a selectively permeable capsule or device, the use of tissue from other species can still result in an immunological reaction. Xenografts are thus more difficult to protect from destruction by the host immune system than allografts (Gill and Wolf, 1995).
  • An additional limitation regarding the use of non-human cells and tissues in transplantation is the hazard of epizootic events: the introduction and propagation of animal pathogens in the human population. Given these concerns, human cells and cell lines are prefe ⁇ ed biological materials for the cell-based delivery of LCAT.
  • the prefe ⁇ ed LCAT-expressing recombinant cells of the invention have the following combination of properties that impart a su ⁇ risingly effective overall therapeutic benefit to the cells. They grow effectively in culture, function effectively in implantable devices in vivo, have a sufficiently stable phenotype, stably secrete LCAT when provided with an exogenous LCAT nucleic acid and are amenable to genetic engineering. These cells are thus culturable, implantable, sufficiently stable, engineerable and stably secrete therapeutic amounts of LCAT. Such cells may express any therapeutically effective amount of biologically effective LCAT, including efficient low doses, doses to restore normal levels and doses to create supra-normal levels.
  • ⁇ G H04 cells The prefe ⁇ ed culturable, implantable, stable, engineerable and stably secreting cells of the invention are exemplified by ⁇ G H04 cells.
  • ⁇ G H04 cells is used to mean the same as BG H04 cells and H04 cells (generally with the LCAT transgene, although also parental). These cells are derived from a human lung carcinoma (ATCC NCI H1299/CRL- 5803) and are shown herein to have many advantageous features for use in LCAT delivery.
  • ⁇ G H04 cells shave a homozygous partial deletion of the p53 protein, and lack expression of p53 protein. The cells are able to synthesize the peptide NMB at 0.1 pmol/mg protein, but not the gastrin releasing peptide (GRP).
  • GRP gastrin releasing peptide
  • a variety of host cells are useful as starting cells for engineering to provide low, but therapeutically effective doses of LCAT, as disclosed herein.
  • Virtually all cells secrete proteins through a constitutive, non-regulated secretory pathway. Any such cell may be used in this invention.
  • Secretory cells i.e., cells with a regulated secretory pathway, are evidently suitable and will generally be prefe ⁇ ed for co-expression embodiments. These subsets of cells are able to secrete proteins through a specialized, "regulated secretory pathway". Proteins destined for secretion by either mechanism are targeted to the endoplasmic reticulum and pass through the Golgi apparatus.
  • Constitutively secreted proteins pass directly from the Golgi to the plasma membrane in vesicles, fusing and releasing the contents constitutively without the need for external stimuli.
  • Recombinant LCAT may be secreted by this pathway.
  • proteins leave the Golgi and concentrate in storage vesicles or secretory granules. Release of the proteins from secretory granules is regulated, requiring an external stimulus.
  • This external stimulus defined as a secretagogue, can vary depending on cell type, optimal concentration of secretagogue and dynamics of secretion. Proteins can be stored in secretory granules in their final processed form.
  • secretagogue is thus a substance that stimulates the secretion of a polypeptide via the regulated pathway.
  • Secretagogues can be physiological in nature, e.g., glucose, amino acids, or hormones, or pharmacological, e.g., IBMX, forskolin, or sulfonylureas.
  • a cell specialized for secreting proteins via a regulated pathway also can secrete proteins via the constitutive secretory pathway. Many cell types secrete proteins by the constitutive pathway with little or no secretion through a regulated pathway; all are suitable for use in the invention.
  • secretory cell defines cells specialized for regulated secretion, and excludes cells that are not specialized for regulated secretion.
  • the regulated secretory pathway is found in secretory cell types such as endocrine, exocrine, neuronal, some gastrointestinal tract cells and other cells of the diffuse endocrine system.
  • Regulated secretory cells present a natural bioreactor containing specialized enzymes involved in the processing and maturation of secreted proteins. These processing enzymes include endoproteases (Steiner et al, 1992) and carboxypeptidases (Flicker, 1988) for the cleavage of prohormones to hormones and PAM, an enzyme catalyzing the amidation of a number of peptide hormones (Eipper et al., 1992). Similarly, maturation and folding of peptide hormones is performed in a controlled, stepwise manner with defined parameters including pH, calcium and redox states.
  • Complete processing requires sufficient levels of the processing enzymes as well as sufficient retention of the maturing peptides. In this way, physiological signals leading to the release of the content of the secretory granules ensure release of fully processed, active proteins.
  • Secretory cells for use in this invention include neuroendocrine cells, including, but not limited to, pancreatic ⁇ cells, pancreatic ⁇ cells and pituitary cells.
  • neuroendocrine cells suitable for use in the invention are shown in Table 5 (Pearse and Takor, 1979; Nylen and Becker, 1995).
  • Particularly defined secretory cells useful as host cells in the present invention are shown in Table 6.
  • KEY NCI, National Cancer Institute; ATCC, American Type Culture Collection; NE, neuroendocrine; PAM, peptidylglycine alpha- amidating monooxygenase; SYN, synaptophysin; PC, proconvertase; VIM, vimentin; AB, antibiotic; S/R, sensitive/resistant; G, G418; H, hygromycin; O, ouabain; P, puromycin; B, blasticidin; Hd, histidinol; Mca, mycophenohc acid; Z, Zeocin; TG, transgene expression +/-; NP, neomycin phosphotransferase; I, insulin; G, glucagon/glycentin; GH, growth hormone; NT, not tested
  • the secretory cells employed as starting materials may be primary cells or established cell lines that are engineered only to express LCAT. Alternatively, primary cells or cell lines engineered to include other advantageous features may be employed.
  • the cells may be immortalized, or may be engineered to become immortalized. Any immortalized cell lines that do not retain certain desired properties may be re-engineered to provide such properties.
  • the origin of the starting cells for use in the present invention thus includes human tissues and tumors of neuroendocrine lineages that have a well defined regulated secretory pathway. Cells with defined conditions for culturing ex vivo with some replicative capacity are also prefe ⁇ ed. Pancreatic ⁇ cells, pancreatic ⁇ -cells and pituitary cells may be prefe ⁇ ed in certain embodiments.
  • the islets of Langerhans are scattered diffusely throughout the pancreas. Principally, four polypeptide hormones, insulin, glucagon, somatostatin and pancreatic polypeptide, are synthesized and secreted by these cells. Each of the islet hormones is secreted by a distinct cell type: ⁇ cells secrete glucagon, ⁇ cells secret insulin, ⁇ cells secrete somatostatin and pancreatic polypeptide is secreted by PP-cells. In the context of the present invention, any of such cells may be engineered to express LCAT.
  • more suitable vehicles for use in the present invention may be any one or more of several cell lines derived from islet ⁇ cells that have emerged over the past two decades. While early lines were derived from radiation- or virus-induced tumors (Gazdar et al, 1980, Sante ⁇ e et al., 1981), more recent work has centered on the application of transgenic technology (Efrat et al, 1988; Miyazaki et al, 1990).
  • Insulinoma lines provide an advantage in that they can be grown in essentially unlimited quantity at relatively low cost. Although most exhibit differences in their glucose- stimulated insulin secretory response relative to normal islets, these differences are not unduly limiting in regard to recombinant LCAT production.
  • RIN 1046-38 cells are also derived from a radiation-induced insulinoma and are glucose responsive when studied at low passage numbers (Clark et al, 1990).
  • GLUT-2 and glucokinase are expressed in low passage RIN 1046-38 cells, but are gradually diminished with time in culture in synchrony with the loss of glucose-stimulated insulin release (Ferber et al, 1994).
  • Restoration of GLUT-2 and glucokinase expression in RIN 1046-38 cells by stable transfection restores glucose-stimulated insulin secretion (Ferber et al, 1994), and the use of these genes as a general tool for engineering of glucose sensing is described in U.S. Patent No. 5,427,940.
  • INS-1 insulinoma cell line
  • INS-1 insulinoma cell line
  • ⁇ TC cells Cell lines derived by transgenic expression of T-antigen in ⁇ cells (generally termed ⁇ TC cells) may also be used (Efrat et al, 1988; Miyazaki et al, 1990; Efrat et al, 1993).
  • Miyazaki et al. (1990) isolated two classes of clones from transgenic animals expressing an insulin promoter/T-antigen construct. Glucose-unresponsive lines such as MIN-7 were found to express GLUT-1 rather than GLUT-2 as their major glucose transporter isoform, while MIN-6 cells were found to express GLUT-2 and to exhibit normal glucose-stimulated insulin secretion (Miyazaki et al, 1990).
  • Efrat and coworkers demonstrated that the cell line ⁇ TC-6, which exhibits a glucose- stimulated insulin secretion response that resembles that of the islet in magnitude and concentration dependence, expressed GLUT-2 and contained a glucokinase:hexokinase activity ratio similar to that of the normal islet (Efrat et al, 1993). With time in culture, the major change was a large (approximately 6-fold) increase in hexokinase expression (Efrat et al., 1993). Furthermore, overexpression of hexokinase I, but not GLUT-1, in well-differentiated MIN-6 cells results in both increased glucose metabolism and insulin release at subphysiological glucose concentrations.
  • the culturing of human insulinomas may be performed using the following method.
  • tissue culture media BetaGene medium supplemented with 3.5% fetal bovine serum (FBS), 200 U and ⁇ g/ml pemcillin/streptomycin, and 50 ⁇ g/ml gentamycin.
  • FBS fetal bovine serum
  • the tissue is kept on ice and sterile, keeping the transit time to less than 30 minutes.
  • the tissue is minced with iris scissors until it is reduced to pieces 1 mm or smaller.
  • the tumor is then transfe ⁇ ed to 40 mesh tissue sieve through which the large pieces are forced using rubber pestle.
  • the cells are then washed twice for a period of 15 minutes each with fresh culture media containing antibiotics.
  • the tissue is then split onto standard Falcon tissue culture dishes and dishes coated with matrigel extracellular matrix.
  • the tissue is maintained under standard tissue culture atmospheric conditions of 37°C; 5% CO2/95% air; and humidified.
  • the tissue is then cultured with media composed of 30% conditioned tissue culture media (BetaGene medium containing 3.5% fetal bovine serum (FBS) conditioned by culture with ⁇ G 261/13, a rat ⁇ cell line stably transfected with pCB6 expressing the full length human growth hormone coding region), 70% BetaGene Medium product # 62469-79P, 1% FBS, 50 ⁇ g/ml gentamycin.
  • a human insulinoma (HT6#2) was cultured, and has been found to secrete insulin for over 150 days.
  • Cells from the intermediate lobe may have an advantage, as there is a suggestion that this cell type can survive and sustain secretory function in autoimmune disease.
  • the POMC promoter was used to drive expression of insulin in the cells of the intermediate lobe of transgenic nonobese diabetic (NOD) mice.
  • NOD nonobese diabetic
  • Such cells were resistant to autoimmune-dependent destruction even when implanted next to islets in which ⁇ cells were destroyed during the course of the disease (Lipes et al., 1996). Such cells would therefore be useful in the present invention as they would be less prone to attack from the host.
  • AtT-20 cell A well known example of engineered pituitary cells is the AtT-20 cell, which is derived from ACTH secreting cells of the anterior pituitary. Stable transfection of AtT-20 cells with a construct in which a viral promoter is used to direct expression of the human proinsulin cDNA results in cell lines that secrete the co ⁇ ectly processed and mature insulin polypeptide (Moore et al., 1983). Insulin secretion from such lines (generally termed AtT-20ins) can be stimulated by agents such as forskolin or dibutyryl cAMP, with the major secreted product in the form of mature insulin.
  • agents such as forskolin or dibutyryl cAMP
  • AtT-20 cells express the glucokinase gene (Hughes et al., 1991; Liang et al, 1991) and at least in some lines, low levels of glucokinase activity (Hughes et al., 1991 and 1992; Quaade et al, 1991), but are completely lacking in GLUT-2 expression (Hughes et al, 1991 and 1992).
  • AtT-20ins cells The studies with AtT-20ins cells are important because they demonstrate that neuroendocrine cell lines that normally lack glucose-stimulated peptide release may be engineered for this function.
  • Other cell lines that are characterized as neuroendocrine, but lacking in endogenous glucose response include PC 12, a neuronal cell line (ATCC CRL 1721) and GH3, an anterior pituitary cell line that secretes growth hormone (ATCC CCL82.1).
  • these lines do exhibit other properties important for this invention, such as a regulated secretory pathway, expression of endopeptidases required for processing of prohormones to their mature hormone products, and post-translational modification enzymes.
  • ⁇ G HOI cells are human colorectal carcinoma cells having an epithelial mo ⁇ hology. These cells grow in floating aggregates of round cells.
  • a characteristic that makes these cells useful in the context of the present invention is that they contain cytoplasmic dense core granules characteristic of endocrine secretion.
  • ⁇ G H02 cells are derived from a thyroid medullary carcinoma.
  • ⁇ G H03 cells are derived from a human non-small cell lung carcinoma. These cells have a neuroendocrine phenotype and can be grown in a monolayer. This line was derived from a lung tissue obtained from a patient prior to therapy.
  • ⁇ G H03 cells express a variety of proteins that are characteristic of neuroendocrine cells, including synaptophysin, peptide- amidating enzyme peptidylglycine ⁇ -amidating monooxygenase (PAM), prohormone convertase 1/3, (PC 1/3) and prohormone convertase 2 (PC2).
  • PAM peptide- amidating enzyme peptidylglycine ⁇ -amidating monooxygenase
  • ⁇ G H03 cells are derived from a human non-small cell lung carcinoma. These cells have a neuroendocrine phenotype and can be grown in a monolayer. This line was derived from a lung tissue obtained from a patient prior to therapy.
  • ⁇ G H03 cells express a variety of proteins that are characteristic of neuroendocrine cells, including synaptophysin, peptide-amidating enzyme peptidylglycine ⁇ -amidating monooxygenase (PAM), prohormone convertase 1/3, (PCI/3) and prohormone convertase 2 (PC2).
  • synaptophysin peptide-amidating enzyme peptidylglycine ⁇ -amidating monooxygenase (PAM), prohormone convertase 1/3, (PCI/3) and prohormone convertase 2 (PC2).
  • ⁇ G H03 cells as obtained from the ATCC are not able to synthesize the peptide neuromedin B (NMB) or the gastrin releasing peptide (GRP).
  • ⁇ G H03 cells are sensitive to antibiotics, a useful property for the present invention.
  • ⁇ G H05 (ATCC CRL-5808) cells are classic small cell lung cancer cells with an epithelial mo ⁇ hology.
  • ⁇ G H06 (ATCC CRL-5815), having an epithelial mo ⁇ hology, was derived from tissue taken prior to therapy. This is the best differentiated of the available bronchial carcinoid lines.
  • the cells are able to synthesize the peptide NMB (at 0.1 pmol/mg protein), but not the gastrin releasing peptide (GRP).
  • GFP gastrin releasing peptide
  • the cell line secretes a parathyroid hormone-like protein, which is calcium stimulated through a protein kinase C pathway. Further, growth of NCI-H727 cells is inhibited by epidermal growth factor (EGF) receptor monoclonal antibodies.
  • EGF epidermal growth factor
  • ⁇ G H07 (ATCC CRL-5804) cells were derived from cells recovered from pleural effusion obtained from a patient prior to therapy.
  • ⁇ G H08 are carcinoma cells isolated from a stage 3A squamous cell lymph node carcinoma (ATCC CRL-5867).
  • ⁇ G H09 are derived from an atypical lung carcinoid and are available as ATCC CRL-5838.
  • the ⁇ G H10 cell line is a commercially available cell line derived from lung carcinoma ATCC Number CCL-185.
  • Another similar cell line is ATCC number CCL-185.1, derived from CCL-185, which was initiated through explant culture of lung carcinomatous tissue.
  • CCL-185.1 cells are adapted to growth in serum-free medium.
  • ⁇ G Hl l cells may be obtained form ATCC HTB-9 and are derived from a bladder carcinoma.
  • ⁇ G HI 3 (ATCC CRL-2139) cells are from a primitive neuroectodermal brain tumor.
  • ATCC CCL-249 cells are designated herein as ⁇ G HI 4 and are derived from a colon adenocarcinoma.
  • ⁇ G HI 5 cells are from a colorectal carcinoma (ATCC CCL-253) and have an epithelial phenotype. This line was derived from a metastasis to the abdominal wall obtained from a patient after treatment with 5-fluorouracil.
  • ⁇ G HI 6 are commercially available as ATCC CRL-5974. These are gastric carcinoma cells.
  • ATCC HTB-10 cells are refe ⁇ ed to herein as ⁇ G HI 8, these cells are derived from a neuroblastoma cell line and is one of two cell lines (see also ATCC HTB-11) of neurogenic origin.
  • ⁇ G H19 (ATCC HTB-184) cells are small cell lung carcinoma cells of an extrapulmonary origin and are from an adrenal metastasis in an adult.
  • ⁇ G H20 (ATCC HTB-177) cells are a large cell carcinoma cell line derived from the pleural fluid of a patient with large cell cancer of the lung.
  • the cells stain positively for keratin and vimentin but are negative for neurofilament triplet protein.
  • the line expresses some properties of neuroendocrine cells, is relatively chemosensitive and can be cloned in soft agar (with or without serum).
  • ⁇ G H21 (ATCC CRL-2195) is yet another small cell lung carcinoma cell that may be useful as a starting cell in the present invention. It can grow as suspension and loosely adherent culture and is a biochemically stable continuously cultured cell line. The line was derived from a non-encapsulated primary lung tumor from the apical portion of the upper lobe of the left lung. This cell line is an unusual undifferentiated large cell variant of small cell lung carcinoma. It has the mo ⁇ hology of a variant, but the biochemical properties of a classic SCLC. The cells have neuroendocrine markers L-dopa decarboxylase and dense core secretory granules.
  • ⁇ G H23 is a long-term tissue culture cell line derived from a metastatic human carcinoid tumor of the pancreas (Evers et al, 1991; Parekh et al, 1994). This cell line also is known as BON (Evers et al, 1991). Tumors derived from this cell line are histologically identical to the original tumor. The cells have significant amounts of neurotensin, pancreastatin, and serotonin (5-HT), demonstrated in the cells by radioimmunoassay (RIA) and the presence of chromogranin A, bombesin, and 5-HT is confirmed by immunocytochemistry.
  • RIA radioimmunoassay
  • the cells possess neurosecretory granules and functional receptors for acetylcholine, 5-HT, isoproterenol, and somatostatin.
  • BON cells possess a specific transport system for uptake of 5-HT from the medium; this uptake system may be a route for regulation of autocrine effects of 5-HT on carcinoid cells (Parekh et al., 1994).
  • This unique human carcinoid tumor cell line provides an exemplary starting material for the bioengineering described herein and is useful in that it possesses intracellular mechanisms ideally adapted for secretagogue action in the release of amines and peptides.
  • ⁇ G H25 (ATCC HB-8065), derived from a hepatoblastoma.
  • This cell line produces ⁇ -fetoprotein; albumin; ⁇ 2- macroglobulin; ⁇ l-antitrypsin; transfe ⁇ in; ⁇ l-antichymotrypsin; haptoglobin; ceruloplasmin and plasminogen and demonstrates decreased expression of apoA-I mRNA and increased expression of catalase mRNA in response to gramoxone (oxidative stress) complement (C4); C3 activator; fibrinogen; ⁇ -1 acid glycoprotein; ⁇ 2-HS-glycoprotein; ⁇ -lipoprotein; and retinol binding protein.
  • C4 oxidative stress complement
  • cells from resected neuroendocrine tumors may be obtained from explanted tumor samples, such as insulinomas and pituitary tumors.
  • explanted tumor samples such as insulinomas and pituitary tumors.
  • Two exemplary insulinomas have been reported (Gueli et al, 1987; Cavallo et al., 1992).
  • primary human neuroendocrine secretory cells may be engineered to secrete LCAT.
  • Human fetal pancreases at 18 to 24 gestational weeks can be obtained through nonprofit organ procurement centers, with patient consent for tissue donation being obtained. Dissection of specific organs from the fetuses is often done at the procurement centers. Isolation of fetal pancreases and islets is performed by established techniques (Otonkoski et al, 1993; inco ⁇ orated herein by reference).
  • Human organs can also be obtained from autopsies through nonprofit organ procurement centers.
  • High quality human islets are available, for example, from Dr. Camillo Ricordi of the University of Miami Medical Center, an islet transplant surgeon who supplies human islets to scientists throughout the United States. Automated methods for isolation of human pancreatic islets have been established (Ricordi et al., 1988; inco ⁇ orated herein by reference).
  • the expression construct In order to effect expression of the LCAT transgene, the expression construct must be delivered into the chosen cell.
  • the genetic constructs expressing LCAT will preferably be stably integrated into the genome of the host cell or stably maintained in the cell as a separate, episomal segment of DNA.
  • Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of, or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. All expression constructs and delivery methods are contemplated for use in the context of the present invention.
  • the expression construct may consist only of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any method that physically or chemically permeabilizes the cell membrane, such as those described below.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rea ⁇ angement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an expression construct complexed with Lipofectamine (Gibco BRL).
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al., 1987).
  • Wong et al (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
  • Melloul et al. (1993) demonstrated transfection of both rat and human islet cells using liposomes made from the cationic lipid DOTAP, and Gainer et al. (1996) transfected mouse islets using Lipofectamine- DNA complexes.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome- encapsulated DNA (Kaneda et al, 1989).
  • HVJ hemagglutinating virus
  • the liposome may also be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991), or complexed or employed in conjunction with both HVJ and HMG-1.
  • HMG-1 nuclear non-histone chromosomal proteins
  • Expression constructs may be introduced into cells via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high- voltage electric discharge. Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al, 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al, 1986) in this manner. Examples of electroporation of islets include Soldevila et al. (1991) and PCT application WO 91/09939.
  • Expression constructs can also be introduced to cells using calcium phosphate precipitation.
  • Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique.
  • mouse L(A9), mouse C127, CHO, CV1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al, 1990).
  • Expression construct delivery into cells using DEAE-dextran followed by polyethylene glycol is also possible. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985). 4. Particle Bombardment
  • Expression constructs can further be introduced into cells by direct microinjection or sonication loading.
  • Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985), and LTK- fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987).
  • Receptor-mediated delivery vehicles may also be used to deliver expression constructs to cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention. Specific delivery in the context of another mammalian cell type is described by Wu and Wu (1993).
  • Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a DNA-binding agent. Others comprise a cell receptor-specific ligand to which the DNA construct to be delivered has been operatively attached.
  • Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987, 1988; Wagner et al, 1990; Ferkol et al., 1993; Perales et al, 1994; Myers and White, 1996; EPO 0273085; each specifically inco ⁇ orated herein by reference), which establishes the operability of the technique.
  • the ligand will be chosen to co ⁇ espond to a receptor specifically expressed on the secretory cell or neuroendocrine target cell population.
  • the DNA delivery vehicle component of a cell-specific gene targeting vehicle may comprise a specific binding ligand in combination with a liposome.
  • the nucleic acids to be delivered are housed within the liposome and the specific binding ligand is functionally inco ⁇ orated into the liposome membrane.
  • the liposome will thus specifically bind to the receptors of the target cell and deliver the contents to the cell.
  • Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.
  • EGF epidermal growth factor
  • the DNA delivery vehicle component of the targeted delivery vehicles may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding.
  • a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding.
  • Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, inco ⁇ orated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a further example of receptor mediated transfection is adenoviral assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994), and the inventors contemplate using the same technique to increase transfection efficiencies, e.g., into human islets.
  • adenoviral expression vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
  • Adenovirus expression vectors thus include constructs containing adenovirus sequences sufficient to support packaging of the construct and to ultimately express an LCAT construct that has been cloned therein.
  • Exemplary viral vectors including adenoviral vectors that express LCAT are described in PCT patent application WO 96/28553, specifically inco ⁇ orated herein by reference.
  • the expression vectors typically comprise a genetically engineered form of adenovirus.
  • retrovirus the adenoviral transduction of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • TPL 5-tripartite leader
  • recombinant adenowirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is important to isolate a single clone of virus from an individual plaque and examine its genomic structure. Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977).
  • adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about an extra 2 kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone.
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • the prefe ⁇ ed helper cell line is 293.
  • Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 ⁇ m, the cell viability is estimated with trypan blue.
  • Fibra-Cel microca ⁇ iers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the prefe ⁇ ed starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present invention is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce LCAT at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
  • the LCAT polynucleotide may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992), vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992) and gene therapy (Stratford-Pe ⁇ icaudet and Pe ⁇ icaudet, 1991; Stratford- Perricaudet et al, 1990; Rich et al, 1993).
  • Recombinant adenovirus and adeno-associated virus can both infect and transduce non-dividing human primary cells. In fact, gene transfer efficiencies of approximately 70% for isolated rat islets have been demonstrated (Becker et al, 1994a; 1994b; 1996). 8. AAV Transduction
  • Adeno-associated virus is an attractive vector system for expressing LCAT in human cells as it has a high frequency of integration and it can transduce and infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture (Muzyczka, 1992).
  • AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin, et al., 1986; Lebkowski, et al, 1988; McLaughlin, et al., 1988), which means it is applicable for use with human neuroendocrine cells. Details concerning the generation and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each inco ⁇ orated herein by reference.
  • AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt et al., 1994; Lebkowski et al, 1988; Samulski et al, 1989; Shelling and Smith, 1994; Yoder et al., 1994; Zhou et al, 1994; Hermonat and Muzyczka, 1984; Tratschin et al, 1985; McLaughlin et al, 1988) and genes involved in human diseases (Flotte et al, 1992; Luo et al, 1994; Ohi, et al, 1990; Walsh, et al, 1994; Wei, et al, 1994).
  • An AAV vector has been approved for phase I human trials for the treatment of cystic fibrosis.
  • AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus or a member of the he ⁇ es virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992).
  • another virus either adenovirus or a member of the he ⁇ es virus family
  • helper virus the wild type AAV genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al, 1990; Samulski et al, 1991).
  • rAAV is not restricted to chromosome 19 for integration unless the AAV Rep protein also is expressed (Shelling and Smith, 1994).
  • recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al, 1988; Samulski et al, 1989; each inco ⁇ orated herein by reference) and an expression plasmid containing the wild type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al, 1991; inco ⁇ orated herein by reference).
  • the cells are also infected or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function.
  • rAAV virus stocks made in such fashion are contaminated with adenovirus that must be physically separated from the rAAV particles (for example, by cesium chloride density centrifugation).
  • adenovirus vectors containing the AAV coding regions or cell lines containing the AAV coding regions and some or all of the adenovirus helper genes can be used (Yang et al, 1994; Clark et al, 1995). Cell lines carrying the rAAV DNA as an integrated provirus can also be used (Flotte et al, 1995).
  • Retroviral Transduction Further viral vectors for use with the invention are retroviral vectors.
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • Retroviral vectors are able to transduce and infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome.
  • nucleic acid encoding LCAT is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication- defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983).
  • the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).
  • Packaging cell lines that decrease the likelihood of recombination will preferably be used (Markowitz et al., 1988; Hersdorffer et al, 1990).
  • the media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer.
  • viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden,
  • CAT chloramphenicol acetyltransferase
  • LCAT nucleic acids can also be housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell.
  • a novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
  • adenovirus or AAV transduction of primary cells leading to in vitro expansion of a primary cell population that is then amenable to stable gene transfer by methods requiring cell growth, such as retroviral transduction, plasmid transfection of expanding cells (Lipofectin or electroporation), or a second round of Adenovirus and/or AAV transduction.
  • retroviral transduction plasmid transfection of expanding cells (Lipofectin or electroporation)
  • a second round of Adenovirus and/or AAV transduction is also contemplated, particular as propagation of AAV is dependent upon adenovirus, and using both viruses may lead to more productive transductions.
  • Such a method may increase the number of final cells that have LCAT genes integrated and expressed.
  • adenoviral gene delivery affords a low rate of integration of viral and recombinant DNAs into the host cell genome. Consequently, adenoviral gene expression is diluted when the cells divide.
  • An advantage that adenoviral gene delivery has over many other viral vectors is that entry of the virus into the cell and the expression of transgenic proteins is not dependent on cellular replication. This benefit of adenoviral gene delivery is in contrast to retroviruses, where the integration and sustained expression of virally introduced DNA is dependent on cellular replication. The coupling of these two viral systems thus minimizes the limitations of each and maximally exploits their distinct biological properties.
  • Expression constructs and vectors generally include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In a most general sense, the transcript may be translated into a protein, but it need not be. Antisense and ribozymes that may be used in combination with the present invention are suitable examples. However, where LCAT expression vectors are concerned, it is important that "expression” includes both transcription and translation of the mRNA into the LCAT protein product.
  • the invention may use endogenous cellular promoters to drive LCAT expression.
  • endogenous cellular promoters i.e. promoter trap
  • directed integration of LCAT cDNA using "knock-out" technology.
  • the nucleic acid encoding LCAT or other gene products is under transcriptional control of a promoter.
  • a “promoter” is a DNA sequence recognized by the transcription regulatory machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • the phrase "under transcriptional control” means that the promoter is in the co ⁇ ect location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter is used to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a
  • TATA box such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
  • the particular promoter that is employed to control the expression of LCAT or other nucleic acids is not believed to be important, so long as it is capable of expressing the nucleic acid in the target cell.
  • Suitable promoters include human and viral promoters.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the gene of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters that are well-known in the art to achieve expression of a gene of interest is also contemplated, provided that the levels of expression are sufficient for a given pu ⁇ ose.
  • Table 7 and Table 8 list several promoters and elements that may be employed in the context of the present invention to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression, but merely to be exemplary thereof.
  • the promoter may be in the form of the promoter that is naturally associated with the LCAT gene, as may be obtained from the known genomic sequence and/or by isolating the 5' non-coding sequences located upstream of the coding segment using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein (PCRTM technology is disclosed in U.S. Patent Nos. 4,683,202 and 4,682,195, each inco ⁇ orated herein by reference).
  • Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • Table 7 and Table 8 list viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that can be used in combination with the present invention. Additionally, any promoter/enhancer combination, e.g., any in the Eukaryotic Promoter Data Base EPDB, can also be used. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. This is also encompassed within the invention.
  • Promoters can be further classified into ubiquitous vs. tissue- or cell-specific. Ubiquitous promoters activate transcription in all or most tissues and cell types. Examples of ubiquitous promoters are cellular promoters like the histone promoters, the ubiquitin promoter itself, promoters for many metabolic enzyme genes such as hexokinase I and glyceraldehyde-3- phosphate dehydrogenase, and many viral promoters such as CMVp and the Rous sarcoma virus promoter (RSVp). The use of a ubiquitous promoter in the present invention is contemplated, as such promoters are often strong promoters and as the ex vivo production of engineered cells is already rendered highly specific by the initial cell selection.
  • ubiquitous promoters are cellular promoters like the histone promoters, the ubiquitin promoter itself, promoters for many metabolic enzyme genes such as hexokinase I and glyceraldehyde-3- phosphate dehydrogena
  • Tissue- or cell-specific promoters activate transcription in a restricted set of tissues or cell types or, in some cases, only in a single cell type of a particular tissue.
  • stringent cell-specific promoters are the insulin gene promoters, which are expressed in only a single cell type (pancreatic ⁇ cells) while remaining silent in all other cell types, and the immunoglobulin gene promoters, which are expressed only in cell types of the immune system.
  • tissue-specific promoters are shown above in Table 5 (Pearse and Takor, 1979; Nylen and Becker, 1995) and are exemplary of the types of promoters that may be used in the present invention. Additional promoters will be readily known to those of skill in the art.
  • rat insulin gene promoters include the rat insulin gene promoters, RIP1 (GenBank accession number J00747) and RIP2 (GenBank accession number
  • HIP human insulin promoter
  • HIP can direct cell-specific expression of linked genes in rodent ⁇ cell lines and rat primary islets, albeit, at a somewhat lower level than that observed for
  • RIP1 (Melloul et al, 1993). Further representatives for use in the invention are the glucagon promoter (GenBank accession number X03991); growth hormone promoter (GenBank accession numbers J03071 and
  • Promoters can be modified in a number of ways to increase their transcriptional activity. Multiple copies of a given promoter can be linked in tandem, mutations that increase activity may be introduced, single or multiple copies of individual promoter elements may be attached, parts of unrelated promoters may be fused together, or some combination of all of the above can be employed to generate highly active promoters. All such methods are contemplated for use in connection with the present invention.
  • German et al. (1992) mutated three nucleotides in the transcriptionally important FLAT E box of the rat insulin I gene promoter (RIP), resulting in a three- to four-fold increase in transcriptional activity of the mutated RIP compared to that of a nonmutated RIP as assayed in transiently transfected HIT cells. Also, the introduction of multiple copies of a promoter element from the E. coli tetracycline resistance operon promoter were introduced into the CMV promoter, significantly increasing the activity of this already very potent promoter (Liang et al., 1996).
  • CMV promoter which has high but short-lived transcriptional activity in dog myoblasts
  • MCKp muscle-specific creatine kinase promoter
  • modified rat insulin promoters containing multimerized enhancer elements have been engineered.
  • modRIP contains six multimerized repeats of a 50 base pair region of the cis acting enhancer of RIP, placed upstream of an intact copy of RIP.
  • CMVp Cytomegalovirus promoter
  • CMVp is one of the strongest activating promoters known, but in a very non-tissue specific manner. Therefore, the present modified rat insulin promoters can be used to direct the tissue specific expression of transforming genes at levels presently achievable only with the nonspecific CMVp.
  • a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed.
  • Also contemplated as an element of an LCAT expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the delivery of a nucleic acid in a cell may be identified in vitro or in vivo by including a marker in the expression construct.
  • the marker results in an identifiable change to the transfected cell permitting easy identification of expression.
  • "positively selectable markers” are used.
  • a drug selection marker aids in cloning and in the selection of transformants, for example, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol.
  • tk simplex virus thymidine kinase
  • CAT chloramphenicol acetyltransferase
  • Immunological markers also can be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art. 6.
  • a prefe ⁇ ed embodiment of the in vivo delivery of LCAT via transplantation of engineered cells is the installation of a mechanism that allows for the transplanted cells to be "turned-off ' in both secretory function and growth potential.
  • Scenarios where this "off switch" may need to be employed include a malfunction in the graft, an alteration in the physiology of the host creating an incompatibility with the graft, or a breach in the encapsulation device rendering it permeable to cells.
  • an "off switch" for the transplanted cells will be non-invasive to the host; easy to administer; have short-term, immediate effects; and be selective for the grafted cells and nontoxic to the host.
  • One "off switch” that can fulfill these criteria is the installation of a negative selection system into the transplanted cells.
  • the cells would be engineered to express a protein (negatively selectable marker) that converts a non-toxic substance to a cytotoxic one, through catalysis, transport, or binding.
  • Negatively selectable markers thus confer “cytotoxic sensitivity” and allow one to select or delete genetically altered cells within a mixed population. They differ from “positively selectable markers”, which confer a survival advantage. Negatively selectable markers therefore cause the death of cells that express the marker.
  • Negative selection is used herein in reference to negative selection genes, proteins and systems, also known as “negatively selectable marker genes and proteins” and commonly refe ⁇ ed to as “suicide genes”. The present inventors prefer the terminology "negative selectable marker for targeted cell killing", but all such terms are readily understood in the art.
  • negative selectable markers or cell suicide systems currently in use involve expression of enzymes or transporters. Any one or more of such negative selectable markers may be used for targeted cell killing in the context of the present invention.
  • enzyme systems include he ⁇ es simples virus thymidine kinase (HSV tk) in combination with gancyclovir; cytosine deaminase (CD) in combination with 5-fluorocytosine; and nitroreductase. CD catalyzes the deamination of cytosine to uracil. This enzyme is characteristically expressed in bacteria and fungi.
  • cytosine deaminase expression confers killing in the presence of the compound 5-fluorocytosine, a fluorinated cytosine analogue.
  • the CD enzyme converts 5-fluorocytosine (5-FC) to the toxin 5-fluorouracil (5-FU) by deamination.
  • Mammalian cells are killed by 5-FU, but not 5-FC, as they do not normally express cytosine deaminase or deaminate 5-FC.
  • 5-FC is essentially nontoxic to mammalian cells at concentrations up to 1 mM.
  • Providing CD as a transgene thus allows for selective killing.
  • the presence and expression of the gene has no apparent deleterious effects upon the cells if they are not exposed to 5-FC. However, when such cells are exposed to 5-FC, they cease proliferation and die. The toxicity is due to the deamination of 5-FC to 5-FU by the cells. Normal cells are not inhibited by 5-FC, and only those cell lines with demonstrable cytosine deaminase activity are sensitive to 5- FC toxicity. Further cytosine deaminase killing systems are described in U.S. Patent Nos. 5,358,866 and 5,624,830, each specifically inco ⁇ orated herein by reference, which may be used with the present invention.
  • An example of a negative selection system using a facilitative transporter is the glucose transporter, type 2 (GLUT-2) in combination with streptozotocin (STZ).
  • Transport of the STZ toxin is specific to the GLUT-2 isoform of glucose transporters.
  • Transport provides the intracellular concentrations required to achieve toxicity, particularly at low extracellular concentrations.
  • U.S. patent application Serial No. 08/546,934 and PCT patent application Serial No. WO 97/15668 are specifically inco ⁇ orated herein by reference for pu ⁇ oses of further describing and enabling the use of methods and compositions comprising GLUT-2 and GLUT-2 chimeras in negative selection techniques.
  • Another transporter, recently functionally identified, is ABC1. The transporter, when overexpressed (such as with an inducible promoter), results in depletion of intracellular cholesterol and cell death.
  • the LCAT-expressing cells of the present invention may be further engineered in any of a number of ways. These include modifications to increase LCAT expression and modifications to modulate LCAT expression in response to various signals. The co-expression of other therapeutic proteins is also contemplated.
  • the host cells may also be engineered in areas removed from secretion, e.g., to improve stability, decrease antigenicity or immunogenicity, or such like. Prior to in vivo administration, the cells will also typically be engineered with a "suicide" or negatively selectable marker gene as a safety mechanism. All such additional engineering steps may be considered as "iterative engineering".
  • Non-engineered RIN 1046-38 cells express rat insulin, rather than human insulin. This is overcome by provision of a human insulin gene. The rat insulin gene can be knocked out if desired.
  • the low insulin content of RIN 1046-38 cells one-tenth that of normal human islet ⁇ cells (Clark et al, 1990), needs to be improved. This is also achieved by increasing the insulin content of the cells by further genetic engineered steps.
  • Unengineered RIN 1046-38 cells also lose GLUT-2 and glucokinase expression with time in culture, and hence lose glucose responsiveness (Clark et al, 1990; Ferber et al, 1994).
  • Stable transfection of RIN 1046-38 cells of intermediate, but not high passage, with GLUT-2 reconstitutes glucose-stimulated insulin secretion (GSIS) and induces a 4-fold increase in glucokinase activity relative to untransfected control cells (Ferber et al, 1994).
  • GSIS glucose-stimulated insulin secretion
  • the upregulation of glucokinase activity in response to GLUT-2 transfection is transient though, and the cells lose glucose responsiveness over time (Ferber et al, 1994).
  • the type of "iterative engineering" techniques used to create novel RIN cell lines with stable expression of human insulin, GLUT-2 and glucokinase, and improved GSIS, may be applied to increase LCAT expression from any chosen recombinant cell.
  • LCAT secretion does not need to be precisely regulated, minute-by-minute, in response to a secretagogue such as glucose, the genetic engineering techniques for application to the present invention will be seven simpler than those used to secrete insulin from RIN cells.
  • IRES elements are used to create multigene or polycistronic messages.
  • IRES elements are able to bypass the ribosome scanning model of 5-methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picanovirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements.
  • this includes genes for other secreted proteins, cell surface receptors and selectable marker genes. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
  • an endogenous gene product in a host cell.
  • the targeted endogenous gene encodes a protein normally secreted by the host cell
  • blocking expression of this endogenous gene product while engineering high level expression of LCAT, represents a unique way of usu ⁇ ing secretory function cells for exogenous protein production.
  • Cells generated by this two-step process can be used to express LCAT as a heterologous protein for in vivo cell-based delivery and/or in vitro large-scale production with little or no contaminating or unwanted endogenous protein production.
  • constructs are designed to homologously recombine into particular endogenous gene loci, rendering the endogenous gene nonfunctional.
  • constructs are designed to randomly integrate throughout the genome, resulting in loss of expression of the endogenous gene.
  • constructs are designed to introduce nucleic acids complementary to a target endogenous gene. Expression of RNAs co ⁇ esponding to these complementary nucleic acids will interfere with the transcription and/or translation of the target sequences.
  • constructs are designed to introduce nucleic acids encoding ribozymes, RNA-cleaving enzymes, which will specifically cleave a target mRNA co ⁇ esponding to the endogenous gene.
  • endogenous gene can be rendered dysfunctional by genomic site directed mutagenesis.
  • Each of these methods for blocking protein production is well known to those of skill in the art.
  • PCT Application Serial No. WO 97/26334 and WO 97/26321 describe these methodologies and are specifically inco ⁇ orated herein by reference.
  • Cre/Lox Nonetheless, where multiple rounds of iterative engineering are undertaken, the separate construction events, such as introducing heterologous genes, blocking expression of endogenous gene products by molecular methods (including targeting of both copies of the endogenous gene), and further modification of the host cell to achieve high level expression, will be facilitated by the use of distinct selectable markers.
  • selectable markers for use in mammalian cells is not a limitation to the invention, the Cre/Lox site-specific recombination system (Sauer, 1993, available through Gibco/BRL, Inc., Gaithersburg, Md.) may be used to remove initial drug selection markers out of the genome, thus increasing the number of rounds of engineering possible.
  • the system involves the use of a bacterial nucleotide sequence knows as a LoxP site, which is recognized by the bacterial Cre protein.
  • the Cre protein catalyzes a site- specific recombination event. This event is bidirectional, i.e., Cre will catalyze the insertion of sequences at a LoxP site or excise sequences that lie between two LoxP sites.
  • Cre will catalyze the insertion of sequences at a LoxP site or excise sequences that lie between two LoxP sites.
  • introduction of the Cre protein, or a polynucleotide encoding the Cre protein, into the cell will catalyze the removal of the selectable marker.
  • This technology renders the same selectable marker available for use in further genetic engineering of the cell and is explained in detail in U.S. Patent No. 4,959,317, which is hereby inco ⁇ orated by reference in its entirety.
  • the iterative engineering contemplated within the present invention particularly includes the provision of further recombinant genes expressing products useful in overall cellular processes such as secretion.
  • the genes and proteins provided may be those either not normally associated with the chosen host cell, or those selected for over-expression.
  • Two general classes of recombinant genes for co-expression with LCAT can be defined: those also encoding secreted products and those encoding proteins that remain associated with the host cell in any of a variety of destinations.
  • proteins may be co-expressed with the LCAT of the present invention.
  • many proteins do not have a single sequence, but rather exist in many forms. These forms may represent allelic variations or acceptable mutant forms of a given protein.
  • full length proteins need not always be expressed, with the expression of protein fragments being acceptable in many situations. For instance, in some cases, it may be desirable to express a particular functional domain, e.g., where the protein fragment remains functional but is more stable, or less antigenic, or both. If desired, various proteins may also be expressed as "fusion proteins". Fusion proteins are generated by linking together the coding regions for two proteins, or parts of two proteins.
  • Fusions may be useful in producing secreted forms of proteins that are not normally secreted or producing molecules that are immunologically tagged. Tagged proteins may be more easily purified or monitored using antibodies to the tag.
  • Cells engineered to co-express other proteins with LCAT can be used for either in vitro production of the protein or for in vivo, cell-based therapies.
  • In vitro production entails purification of the expressed protein from either the cell pellet for proteins remaining associated with the cell or from the conditioned media from cells secreting the engineered protein.
  • cell-based therapies are based on secretion of the protein.
  • Recombinant cells that co-express and secrete LCAT along with at least one other therapeutic component can be used to treat animals or humans with various disorders and/or particularly aggravated forms of certain disorders.
  • the provision of genes expressing products that further assist in the control of cholesterol metabolism and/or treat the underlying disease in a supplementary or complementary manner are particularly contemplated, although this is by no means exclusive.
  • Apolipoprotein A-I (Apo A-I) is a major co-factor for LCAT function and is contemplated for combined use with the invention. If desired, those of ordinary skill in the art will be able to select a particular patient population that would most benefit from combination therapy with Apo A-I and LCAT.
  • LCAT lipase
  • HL hepatic lipase, Genbank Accession Nos. AF037404, PI 1150 and P07098).
  • Estrogens and estrogen analogs may further be administered to increase the serum level of LCAT activity by up-regulating any existing endogenous production of LCAT.
  • cholesterol metabolism and LCAT impact vascular and heart diseases patients to be treated by the present invention will include patients having or at risk for developing vascular disease and heart problems. Accordingly, it may be desirable to increase blood flow to occluded vessels, particularly those within the heart. Accordingly, angiogenic factors may be co- administered with the LCAT of the present invention. A wide variety of such factors are known, of which VEGF is a cu ⁇ ently prefe ⁇ ed example for use herewith.
  • peptide hormones may be co-expressed with LCAT in this invention.
  • Peptide hormones may be grouped into three classes, defined by the complexity of their post- translational processing. Class I is represented by growth hormone, prolactin and parathyroid hormone and the peptides shown in Table 9. These require relatively limited proteolytic processing followed by storage and stimulated release from secretory granules. Leptin may be prefe ⁇ ed for use with the present invention in that leptin-induced weight loss may contribute to general health and avoiding diet-related complications, including heart attack.
  • Class II is represented by insulin and glucagon and the peptides shown in Table 10. Further proteolytic processing is required, with both endoproteases and carboxypeptidases processing of larger precursor molecules occu ⁇ ing in the secretory granules. Table 10 Class II Human Peptide Hormones
  • GIP Gastric Inhibitory Peptide
  • Class III is represented by amylin, glucagon-like peptide I and calcitonin and the peptides shown in Table 11.
  • amidation of the C-terminus is required for proper biological function.
  • Any one or more peptide hormones from of any class may be co-expressed with LCAT according to the present invention.
  • Glucoregulatory hormones include insulin, glucagon, epinephrine, and cortisol. Insulin is the major hormone responsible for lowering plasma glucose concentrations. Glucose raising or "glucose counter-regulatory hormones" include glucagon (X03991, J04040), epinephrine, norepinephrine, pancreatic polypeptide, vasopressin (AF032388, U04357, L22206) and cortisol.
  • One or more other proteins that are normally secreted can be engineered into recombinant cells for co-expression with LCAT.
  • Useful human proteins include soluble CD4, Factor VIII, Factor IX, von Willebrand Factor, TPA, urokinase, hirudin, interferons, TNF, interleukins, hematopoietic growth factors, antibodies, albumin, transfe ⁇ in and nerve growth factors.
  • proteins not normally secreted by a cell, can also be engineered into recombinant cells for co-expression and secretion with LCAT. These proteins are those that are normally maintained within a cell, but have been modified such that they are now secreted by the cell. Suitable modifications include creating novel fusion proteins using signal peptides, site-directed mutagenesis or even expression of truncated, engineered proteins, each of which result in the secretion of a normally non-secreted protein (Ferber et al., 1991; Mains et al., 1995).
  • Engineered cells may also be engineered to co-express enzymes of therapeutic value with LCAT.
  • enzymes include, by are not limited, to adenosine deaminase (e.g., Genbank Accession Nos. P55265, U18121, U73107, Z97053, P00813, U75503, DUHUA); galactosidase (e.g., Genbank Accession Nos. P54803, P51569, P23780, D00039); glucosidases (e.g., Genbank Accession Nos. P29064 ( ⁇ -glucosidase) and P26208 ⁇ -glucosidase)); factor IX (e.g., Genbank Accession Nos.
  • sphingolipase lysosomal acid lipase (e.g., Genbank Accession Nos. P38571; S41408); lipoprotein lipase (e.g., Genbank Accession No. P06858); hepatic lipase (e.g., Genbank Accession Nos. AF037404, PI 1150, P07098); pancreatic lipase related protein (e.g., Genbank Accession Nos. P54315, P54317); pancreatic lipase (P16233) and uronidase.
  • lysosomal acid lipase e.g., Genbank Accession Nos. P38571; S41408
  • lipoprotein lipase e.g., Genbank Accession No. P06858
  • hepatic lipase e.g., Genbank Accession Nos. AF037404, PI 1150, P07098
  • Prefe ⁇ ed engineering strategies for use with the present invention are those that enhance, alter or refine the secretory process.
  • the transgenes for joint expression with LCAT generally fall within the second class of recombinant genes; namely, genes that encode proteins and polypeptides that remain associated with the host cell.
  • the additional recombinant proteins may be soluble or membrane associated and may be associated with any of a variety of cellular destinations, including the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, Golgi, membrane of secretory granules and plasma membrane.
  • Genes and cDNAs encoding a number of proteins useful in these aspects of the invention are available. These include cell surface receptors, transporters and channels, such as GLUT-2, CFTR, leptin receptor, sulfonylurea receptor, inward rectifying channels, ⁇ 2-adrenergic receptor, pancreatic polypeptide receptor, somatostatin receptor, glucocorticoid receptor, potassium inward rectifying channel, GLP-1 receptor and muscarinic receptor etc.
  • cell surface receptors such as GLUT-2, CFTR, leptin receptor, sulfonylurea receptor, inward rectifying channels, ⁇ 2-adrenergic receptor, pancreatic polypeptide receptor, somatostatin receptor, glucocorticoid receptor, potassium inward rectifying channel, GLP-1 receptor and muscarinic receptor etc.
  • proteins include protein processing enzymes, such as PC2 and PC3, and PAM; transcription factors, such as IPF1, and metabolic enzymes, such as adenosine deaminase, phenylalanine hydroxylase and glucocerebrosidase.
  • protein processing enzymes such as PC2 and PC3, and PAM
  • transcription factors such as IPF1
  • metabolic enzymes such as adenosine deaminase, phenylalanine hydroxylase and glucocerebrosidase.
  • a glucose-stimulated release of LCAT can be engineered into a host cell.
  • the components required for glucose-stimulated insulin release can also be engineered into host cells designed to co-express insulin and LCAT.
  • host cells designed to co-express insulin and LCAT As diabetes and weight problems are often related, which can impact atherosclerosis and coronary artery disease, the combined secretion of LCAT and insulin may be desirable.
  • GSIS glucose-stimulated insulin secretion
  • One or more glucose-sensing components can be re-engineered into a host cell if required.
  • the basis for engineering cells to produce glucose-regulated insulin secretion is disclosed in U.S. Patent No. 5,427,940, inco ⁇ orated herein by reference, which teaches islet and non-islet cell lines of neuroendocrine origin that are engineered for insulin expression and glucose regulation.
  • U.S. Patent No. 5,427,940 teaches that three functional genes are required to give glucose-responsive insulin secreting capacity in a cell: an insulin gene, a GLUT-2 glucose transporter gene and a glucokinase gene. In re-engineering GSIS, it may be that only one of these three genes needs to be supplied, expressed or overexpressed.
  • glucokinase gene and a GLUT-2 gene may still be used to provide glucose-responsiveness.
  • Those of ordinary skill in the art will be readily able to test the levels of glucokinase and GLUT- 2 expression, either by molecular biological hybridization and/or biochemical activity assays, in order to determine whether they are sufficiently expressed and/or active, or may need to be supplied in recombinant form. If a cell does not express either of the aforementioned genes in a functional fashion, or at physiological levels, both genes could be introduced.
  • the constructs of GenBank accession numbers J03145 and M25807, respectively may be used.
  • any one or more of a variety of methods may be used to inhibit hexokinase to achieve the desired effect, including gene knockout.
  • Trehalose-6-phosphate synthase enzymes and ribozymes may also be used to inhibit hexokinase, as disclosed in U.S. Patent No. 5,854,067, specifically inco ⁇ orated herein by reference.
  • cell surface signaling receptors that potentiate the glucose-stimulated degranulation of ⁇ cells can also be engineered into host cells, including the glucagon-like peptide I receptor (Thorens, 1992) and the glucose-dependent insulinotropic polypeptide receptor (also known as gastric inhibitory peptide receptor) (Usdin, 1993). In addition to GLUT-2 and glucokinase, these ⁇ cell-specific signaling receptors are involved in secretory granule release in response to glucose.
  • the present invention also contemplates augmenting or increasing the capacity of cells to produce biologically active LCAT and other polypeptides. This can be accomplished by the expression or overexpression of proteins involved in maintaining the specialized phenotype of host cells, especially their secretory capacity. In some instances, proteins involved in protein processing may be expressed, such as the endoproteases PC2 and PC3 (Steiner et al, 1992) or the peptide amidating enzyme, PAM (Eipper et al, 1992) in the case of amidated peptide hormones.
  • PC2 and PC3 the endoproteases PC2 and PC3
  • PAM peptide amidating enzyme
  • IPF-1 Insulin Promoter Factor 1
  • pancreatic ⁇ cells pancreatic ⁇ cells
  • IPF-1 Insulin Promoter Factor 1
  • the overexpression of IPF-1 can be used to increase LCAT transgene expression under the control of an insulin enhancer/promoter.
  • IPF-1 may also assist ⁇ cell-based host cells to maintain the required differentiated functions of normal ⁇ cells.
  • Cell surface signaling proteins involved in non-glucose-stimulated release of secretory granule contents can also be engineered into a cell of the invention.
  • Examples include releasing factor receptors such as Growth Hormone Releasing Factor Receptor (Lin et al., 1992) and Somatostatin or Growth Hormone Releasing Hormone Receptor (Mayo, 1992).
  • the co-expression embodiments of the present invention further provide engineered cells with a more fine-tuned secretory response and/or with the capacity to secrete recombinant LCAT in response to new stimuli.
  • These aspects of the invention therefore provide cells from which the secretion of LCAT is more precisely regulated in response to physiological changes and, particularly, is controllable in response to the administration of (exogenous) pharmacological agents.
  • Such enhanced regulation of the secretory response is preferably achieved by the co- expression of LCAT with one or more recombinant receptor proteins that provide or increase the sensitivity of the cell to naturally-occu ⁇ ing and/or pharmacological modulators of secretion.
  • Secretory cells sense a variety of extracellular molecules through metabolism, receptors, and ion channels. Each of these sensing mechanisms impact intracellular calcium levels, which, in turn, control the release of, secreted products. Aside from modulating intracellular metabolism, such as by changing hexokinase-glucokinase ratios, the engineering of novel control mechanisms into secretory cells generally involves the expression or overexpression of one or more cell surface receptors and/or channels. Any receptor or channel that affects membrane polarization and hype ⁇ olarization, alters Ca 2 *i and/or cAMP levels and/or changes protein kinase C activity will impact secretory function and may be used in these aspects of the invention.
  • the principles of enhancing the control of regulated secretion apply to both the stimulation and inhibition of secretion. Modulation of secretion can thus be achieved by installing cell-surface receptors that act as positive or negative regulators of secretion. Further, each class of receptor is subject to activation or inhibition of activity by the binding of receptor- specific ligands, including physiological and pharmacological agents.
  • the receptor stimulates secretion and the ligand activates the receptor, thereby stimulating secretion; the receptor stimulates secretion and the ligand inhibits the receptor, thereby inhibiting secretion; the receptor inhibits secretion and the ligand activates the receptor, thereby inhibiting secretion; and the receptor inhibits secretion and the ligand inhibits the receptor, thereby stimulating secretion.
  • the cell surface proteins that will impact secretory functions in this manner include the following receptors: ⁇ l-adrenergic receptor (M18415, M23533); ⁇ 2-adrenergic receptor (U03866, L31774, U03864, U03865); ⁇ l-adrenergic receptor (U03866, L31774, U03864, U03865); ⁇ 2-adrenergic receptor (J03019); arginine vasopressin receptor (AF032388, U04357, L22206); glucagon-like peptide I receptor ( L23503, U10037, U01156, U01104); somatostatin receptor V (AF004740, L14865, L14856, M81830, M96738, M81829, L07833); SUR channel (L78207, U63455, L78243); KIR channel; pancreatic polypeptide receptor (Z66526, U42387, U42389); muscarinic receptor (X52068
  • Table 12 classifies receptors as positive or negative modulators of secretion and identifies ligands that can act either as inhibitors or activators of secretion.
  • Enhanced regulation of LCAT secretion from engineered cells can thus be achieved by either pre-selecting a cell that expresses the required endogenous receptor and/or by the use of a further transgene that expresses an exogenous form of the desired receptor.
  • LCAT-expressing cells comprising such receptors would be subject to modulation of secretion when administered to animals and patients, including modulation by naturally occurring, physiological agents and modulation in response to pharmacological, or exogenous agents.
  • cell-surface receptors that act as negative regulators of secretion in concert with ligands that stimulate their activity include the following receptor/ligand pairs: ⁇ 2- adrenergic receptor/epinephrine or Clonidine; somatostatin receptor/somatostatin or Octreotide; and glucocorticoid receptor/glucocorticoids. Each of these receptor/ligand pairs thus function to inhibit secretion.
  • SUR sulfonylurea receptor
  • SUR generally functions to increase secretion of insulin from the pancreatic ⁇ cell, and its activity is altered by increases in ⁇ cell glucose metabolism and various post-prandial signals.
  • This receptor can be exploited to decrease secretion when provided with ligands such as diazoxide and the naturally-occurring peptide, alpha endosulfine (Heron et al, 1998). Again, the receptor/ligand pair functions to inhibit secretion.
  • receptor/ligand pairs that function to stimulate secretion.
  • Appropriate mechanisms for regulating LCAT secretion from engineered cells in this manner include the activation of receptors that typically stimulate secretion themselves.
  • the activity of SUR can be modulated through the oral administration of pharmacological compounds such as sulfonylureas or PrandinTM.
  • This form of receptor modulation is already practiced in therapy, wherein the ligands are administered to NIDDM patients, generally timed with food intake, as a means to increase insulin secretion from the endogenous pancreatic ⁇ cell.
  • this invention particularly contemplates using LCAT-secreting cells that express endogenous SUR, or that co-express transgenic SUR, and stimulating secretion from such cells by the administration of a sulfonylurea or PrandinTM.
  • a number of enzymes that are essential for the post-translational modifications have been characterized, with many abundantly expressed in secretory and neuroendocrine cells. Whether manufacturing processes utilizing neuroendocrine cells involve production of purified peptides or cells for implantation, the process should sustain the activity of these enzymes so that bioactive peptides will be produced.
  • Culture media for secretory and neuroendocrine cells have been developed, which optimize both growth and function, and which may be used in the context of the present invention.
  • Such media include components that are important for the activity of enzymes involved in proteolytic processing, such as a stabilized ascorbate (ascorbate-2 phosphate) and trace minerals; components that enhance growth (phosphoethanolamine); components that protect against radical toxicity (ethanolamine/phosphoethanolamine and pyruvate); and other minimal supplements to support secretagogue-induced secretion (serum albumin and transfe ⁇ in, chelators or small molecule ligands).
  • a stabilized ascorbate ascorbate-2 phosphate
  • compositions maintain, and even restore, both the regulated secretory pathway (including normal glucose sensing in human islets) and proteolytic processing in small-scale flask cultures and in bulk-scale production cultures.
  • the compositions are thus useful for maintenance of neuroendocrine phenotype in culturing cell lines, particularly in applications that involve production cultures without serum.
  • the LCAT-expressing cells may be propagated using any one or more of a variety of techniques well known to those of skill in the art. For example, cells may be propagated as non- anchorage dependent cells growing freely in suspension throughout the bulk of the culture; or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation, i.e., a monolayer type of cell growth.
  • the cells may be propagated in a microca ⁇ ier culture.
  • This mode of propagation makes it possible to use this system for cellular manipulations, such as cell transfer without the use of proteolytic enzymes, cocultivation of cells, transplantation into animals, and perfusion of the culture using decanters, columns, fluidized beds, or hollow fibers for microca ⁇ ier retainment.
  • the present invention provides both prophylactic and therapeutic regimens.
  • "Prophylactic treatments" are intended for those subjects at risk for developing atherosclerosis or other conditions associated with LCAT deficiency.
  • One risk factor is an atherogenic lipoprotein profile. For example, a ratio of serum cholesterol to high density lipoproteins of above 5:1 indicates a higher than average risk of developing atherosclerosis.
  • Other factors indicating increased risk for atherosclerosis include a serum cholesterol level of above about 240 mg/dl; a high density lipoprotein level below about 35 mg/dl; and a low density lipoprotein level above about 190 mg/dl.
  • the "therapeutic treatment" aspects of the invention will be applied to individuals already suffering from atherosclerosis or a disease associated with LCAT deficiency, optionally after confirming that the individual is best suited to recombinant cell therapy, e.g., by pre-testing with a diagnostic assay of the present invention.
  • animals, mammals and human subjects with atherosclerosis are individuals of any appropriate animal or mammalian species, or a human subject, that exhibits develops one or more signs of atherosclerosis.
  • the signs or indicators of atherosclerosis include, for example, the development of cholesterol plaques in the arteries and calcification, the extent of which can be determined by Sudan IV staining, or the development of foam cells in an artery.
  • Atherosclerosis also is characterized by a narrowing of the arteries detected by, for example, coronary angioplasty, ultrasound and ultrafast CT.
  • the methods of the present invention involve the use of recombinant cells to increase serum LCAT activity levels in all such individuals in need of prophylaxis or therapy to a biologically or therapeutically effective level.
  • Therapeutic effectiveness depends on several factors, including the species, the manner of administration, the general health of the subject, the desired result (e.g., prophylaxis or therapeutic treatment) and the judgment of the prescribing physician. For example, the practitioner may decide what risk levels for heart disease indicate prophylactic treatment, and what target level of LCAT is indicated for the treatment of a person already suffering from atherosclerosis. Nonetheless, the present invention provides the following guidance as to valuable indicators of therapeutic effectiveness.
  • Suitable measures of therapeutic levels of serum LCAT activity are those effective to increase at least one beneficial clinical parameter or at least partially alleviate at least one indication of disease or clinical symptom in at least a sub-set of patients.
  • a further indication of therapeutic serum LCAT levels is the ability to delay the onset of one or more symptoms towards more advanced age.
  • delayed onset and/or reduced levels of corneal opacity, anemia and proteinuria are further measures of the therapeutic benefits of LCAT.
  • Particularly prefe ⁇ ed measures of therapeutic serum LCAT levels include levels of LCAT activity effective to increase the HDL/LDL ratio and/or decrease the accumulation of cholesterol.
  • An LCAT level sufficient to measurably increase the HDL/LDL ratio and/or to measurably decrease the rate of accumulation of cholesterol is any level that is higher, and preferably, significantly higher, than the LCAT levels in the animal or patient before treatment.
  • LCAT should be provided to achieve a cholesterol esterification rate of at least 200 nmol/ml/hr, and further recommends cholesterol esterification rates of at least 2000 nmol/ml/hr - - twenty times the normal value.
  • WO 97/17434 In terms of LCAT mass, after quoting the normal LCAT protein level as 5 ⁇ g/ml serum, the authors of WO 97/17434 teach the desired therapeutic range to be at least 15 ⁇ g/ml up to at least lOO ⁇ g/ml. These are three to twenty times the normal values. From studies in a rabbit model, WO 97/17434 tends to suggest that the mass quantity of human LCAT in the serum should be increased by about five times above the normal human level.
  • the present invention is founded on the su ⁇ rising realization that the art has grossly over-estimated the levels of LCAT activity required to achieve a meaningful therapeutic benefit in an LCAT deficient patient.
  • the present inventors term the levels suggested in the prior art "super high", and believe them to be inefficient and wasteful (although not necessarily detrimental).
  • the present invention contemplates treating LCAT deficient patients with recombinant cells that secrete LCAT in amounts to restore only sub-wild type levels in patients.
  • restoration to only 5% wild-type level is predicted to have a beneficial effect, in that signs and symptoms of disease are alleviated and/or that onset of disease is slowed.
  • the present inventors' new inte ⁇ retation of the data shows that the homozygous patient with the highest LCAT activity (9.1) fared the best of the comparative symptomatic group. This patient has no anemia. Only the patient with very low LCAT (0.07) had proteinuria, a sign of kidney failure. Overall, the cholesterol and triglycerides levels were much better in the subjects that had over 5% wild type LCAT activity.
  • Recombinant cells preferably recombinant cells in implantable devices, can thus be formulated in appropriate unit doses, i.e., a physically discrete, preferably implantable, unit suitable for use in a subject, each unit containing a predetermined quantity of cells calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen.
  • the cells of the present invention may, i ⁇ espective of the culture method chosen, be used in protein production and as cells for in vitro cellular assays and screens as part of drug development protocols.
  • the engineered cells of the present invention are preferably introduced into animals, including human subjects, to provide LCAT thereto.
  • Various encapsulation techniques are now available that enable the cells to be used both in vitro and in vivo.
  • compositions of the LCAT-expressing cells in a form appropriate for the intended application, which will most usually be within a selectively permeable membrane or device. Nonetheless, the cells will generally be prepared as a composition that is essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • One will generally desire to employ appropriate salts and buffers to render the LCAT- expressing cells suitable for introduction into an animal or patient within their selectively permeable membrane, implantable device or other delivery vehicle.
  • Aqueous compositions of the present invention comprise an effective amount of LCAT-secreting cells dispersed in a pharmaceutically acceptable ca ⁇ ier or aqueous medium, and preferably encapsulated.
  • phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable ca ⁇ ier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like. As used herein, this term is particularly intended to include biocompatible implantable devices and encapsulated cell populations. The use of such media and agents for pharmaceutically active cells is well know in the art. Except insofar as any conventional media or agent is incompatible with the cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be inco ⁇ orated into the compositions.
  • the cell preparations may further contain a preservative to prevent growth of microorganisms.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well-known parameters.
  • prefe ⁇ ed embodiments of the invention employ encapsulation and/or microencapsulation of cells in a biocompatible coating in order to provide the cells to an animal or human in vivo.
  • the cells are retained inside semipermeable membranes or capsular coatings, such as hydrogel or alginate membranes, which protect the contents from immunological responses.
  • semipermeable membranes or capsular coatings such as hydrogel or alginate membranes, which protect the contents from immunological responses.
  • the porous membranes permit the exchange of nutrients, gases, and metabolic products with the bulk medium surrounding the capsule containing the cells.
  • Several encapsulation and microencapsulation methods are available that are gentle, rapid and non-toxic and where the resulting membrane is sufficiently porous and strong to sustain the growing cell mass throughout the term of the culture.
  • Implantation methods employing encapsulation techniques are prefe ⁇ ed for a variety of reasons. Particularly, transplantation of cells into animals by these methods significantly prolongs xenograft survival compared to unencapsulated controls (O'Shea and Sun, 1986; Fritschy et al, 1991a). In addition, encapsulation will prevent uncontrolled proliferation of clonal cells.
  • An alternate approach to encapsulation is to simply inject the cells of the invention into the scapular region or peritoneal cavity of mice or rats, where the cells will form tumors (Sato et al, 1962). Implantation by this approach may circumvent problems with viability or function, at least for the short term, which may be encountered with the encapsulation strategy. This approach will allow testing of the function of the cells in experimental animals, which is a viable use of the present invention, but is not applicable as an ultimate strategy for treating humans. Nonetheless, as a pre-clinical test, this will be understood to have significant utility.
  • Certain methods involve encapsulation with alginate-polylysine-alginate and/or soluble alginate, gelled by droplet contact with a calcium-containing solution.
  • Lim (1982) describes cells concentrated in an approximately 1% solution of sodium alginate, which are forced through a small orifice, forming droplets, and breaking free into an approximately 1% calcium chloride solution. The droplets are then cast in a layer of polyamino acid that ionically bonds to the surface alginate. Finally the alginate is reliquified by treating the droplet in a chelating agent to remove the calcium ions.
  • Other methods use cells in a calcium solution to be dropped into a alginate solution, thus creating a hollow alginate sphere.
  • a similar approach involves cells in a chitosan solution dropped into alginate, also creating hollow spheres.
  • Cells may also be implanted using alginate-polylysine encapsulation techniques (O'Shea and Sun, 1986; Fritschy et al, 1991; each specifically inco ⁇ orated herein by reference).
  • the engineered cells are suspended in 1.3% sodium alginate and encapsulated by extrusion of drops of the cell/alginate suspension through a syringe into CaC12. After several washing steps, the droplets are suspended in polylysine and rewashed.
  • the alginate within the capsules is then reliquified by suspension in 1 mM EGTA and then rewashed with Krebs balanced salt buffer.
  • Microencapsulated cells are easily propagated in sti ⁇ ed tank reactors and, with beads sizes in the range of 150-1500 ⁇ m in diameter, are easily retained in a perfused reactor using a fine-meshed screen.
  • the ratio of capsule volume total media volume can kept from as dense as 1:2 to 1 :10. With intracapsular cell densities of up to IO 8 cells/ml, the effective cell density in the culture is 15 x IO 7 cells/ml.
  • microencapsulation over other processes include the protection from the deleterious effects of shear stresses that occur from sparging and agitation, the ability to easily retain beads for the pu ⁇ ose of using perfused systems, scale up is relatively straightforward and the ability to use the beads for implantation.
  • U.S. Patent No. 4,402,694 also inco ⁇ orated herein by reference, describes a body cavity access device containing a hormone source.
  • the device supplies a hormone to a patient.
  • the device is made of an implantable housing that is placed in the body and has an impermeable extraco ⁇ oreal segment and a semipermeable subcutaneous segment.
  • a hormone source including hormone-producing cells, is then removably positioned in the housing to provide a hormone and/or other peptide supply to the patient.
  • Such a device also can contain a sensor located within the subcutaneous segment and operably associated with a dispenser to release medication into the housing and to the patient.
  • Hydrophilic polymeric chambers for encapsulating biologically active tissue and methods for their preparation are described in U.S. Patent No. 4,298,002, specifically inco ⁇ orated herein by reference.
  • the tissue refers to those essential cellular components of a particular organ that is capable of receiving, modifying or secreting hormones.
  • a device comprising such chamber and such tissue is fabricated and implanted in a living body so that said tissue is permitted normal function without being rejected by the host's immunological system.
  • the viability of the tissue in the device is maintained by a co ⁇ elation of factors including pore size and membrane thickness of the hydrophilic chamber.
  • the implanted device allows the inflow of essential nutrients and gases, and outflow of metabolites and products while simultaneously excluding the ingress of cellular components of the host's immunological system.
  • U.S. Patent No. 5,011,472 specifically inco ⁇ orated herein by reference, describes devices and methods to provide hybrid, modular systems for the constitutive delivery of appropriate dosages of active factor to a subject and, in some instances, to specific anatomical regions of the subject.
  • This system includes a cell reservoir containing living cells capable of secreting an active agent, which is preferably adapted for implantation within the body of the subject and further includes at least one semipermeable membrane, whereby the transplanted cells can be nourished by nutrients transported across the membrane while at the same time protected from immunological, bacterial, and viral assault.
  • the systems further include a pumping means, which can be implantable or extraco ⁇ oreal, for drawing a body fluid from the subject into the cell reservoir and for actively transporting the secreted biological factors from the cell reservoir to a selected region of the subject.
  • the hollow fiber has a porosity that selectively allows passage of substances having a molecular weight of less than about 100,000 Daltons.
  • the semi-solid matrix in which the islets are embedded and suspended is formed of an appropriate supporting material such as alginate or agar.
  • U.S. Patent No. 5,549,675, inco ⁇ orated herein by reference describes additional methods for implanting tissue in a host.
  • the methods comprise creating an implant assembly for holding cells including a wall for forming a porous boundary between the host tissue and the implanted cells in the device and implanting the device and then adding the cells.
  • the pore size of the boundary is such that it is sufficient to isolate the implanted cells from the immune response.
  • U.S. Patent No. 5,545,223 further describes methods of making and using ported tissue implant systems and is therefore inco ⁇ orated herein by reference.
  • U.S. Patent Nos. 5,626,561 and 5,787,900, each specifically inco ⁇ orated herein by reference, describe an implantable containment apparatus for a therapeutic device and methods for loading and reloading the device.
  • the implantable containment apparatus is made of selectively permeable material and can be used to contain a therapeutic device, such as a drug delivery device, a cell encapsulation device, or a gene therapy device.
  • a therapeutic device can be easily placed and replaced in the apparatus without damaging tissues associated with the selectively permeable material of the apparatus.
  • U.S. Patent Nos. 5,843,069 and U.S. Patent No. 5,913,998, each specifically inco ⁇ orated herein by reference, also relate to an implantable containment apparatus of selectively permeable material in which a therapeutic device can be placed and replaced without damage.
  • the materials are particularly laminates comprising a first layer of a porous stretched polytetrafluoroethylene material that is impervious to cellular ingrowth and a second layer of porous stretched polytetrafluoroethylene material that is sufficiently porous to permit growth of vascular tissue from a recipient within the pores of the porous stretched polytetrafluoroethylene material up to, but not through, the first layer.
  • hydrophilic surfaces for use as substrates for the immobilization of bioactive species are described.
  • the hydrophilic surfaces are chemically stable, but provide a variety of chemically functional groups for immobilization.
  • Polymeric surfactants are attached to such surfaces and covalently cross-linked thereon to form a first layer.
  • Hydrophilic polymers are then attached to the first layer to form a second layer. The second layer is used to enhance hydrophilicity and to provide a substrate for immobilizing bioactive species.
  • 5,980,889 (Gore Hybrid Technologies, Inc., Flagstaff, Arizona, U.S.A.), specifically inco ⁇ orated herein by reference, concerns cell encapsulating devices capable of maintaining large numbers of viable cells.
  • the devices contain an inert, substantially cell-free core that displaces cells, a permeable membrane and a zone for maintaining cells.
  • the permeable membrane surrounds the core such that the zone of cells is bounded by the core and the permeable membrane.
  • the cell zone may contain a support for cell attachment and the core may have an outer boundary containing a material that promotes cell adhesion.
  • the cell zone has a thickness designed for high cell viability.
  • rods as implantable devices are particularly prefe ⁇ ed.
  • Patent No. 5,980,889 including those modified to include a wet seal, may be formulated for use as rods.
  • Rods are prefe ⁇ ed as the surface area facilitates proper functioning of the implanted cells.
  • such rods are loaded with cells at a density of million cells per cm of device.
  • the rods are flexible before and after loading, so that the cell-loaded devices may be implanted within an animal or human subject in essentially any format.
  • a rod in linear form may be implanted within a limb.
  • the rods may be coiled to any desired shape and size.
  • prefe ⁇ ed formats include those wherein the cell-loaded rods are adapted to form a patch, as exemplified by patches of about 12 cm square, with a 2 mm thickness.
  • thermoplastics of the membranes in the device are designed so that the device can be sealed with the cells and media contained inside.
  • Such devices have been developed by Gore Hybrid Technologies, Inc. (GHT), Flagstaff, Arizona, U.S.A.
  • implantable immunoisolation devices will preferably be used in forms in which the tissue is protected from immune rejection by enclosure within a semipermeable membrane.
  • tissue is protected from immune rejection by enclosure within a semipermeable membrane.
  • Those of skill in the art will understand device design and performance, as it relates to maintenance of cell viability and function. Attention is to be focused on oxygen supply, tissue density and the development of materials that induce neo vascularization at the host tissue- membrane interface; and also on protection from immune rejection. Membrane properties may even be further adapted to prevent immune rejection, thus creating clinically useful implantable immunoisolation devices.
  • RT-PCRTM was performed on human mRNA (Clontech Laboratories, Inc.) using the Superscript Preamplification System (Life Technologies) followed by amplification with High Fidelity Platinum Taq polymerase (Life Technologies). 1.2 ⁇ g of mRNA was transcribed at 42°C for 50 minutes followed by 40 rounds of PCRTM amplification with denaturation at 94 ° C (30 seconds), annealing at 55°C (30 seconds) and extension at 68 C (2 minutes). The amplification was made specific for human LCAT (Accession number M12625; DNA, SEQ ID NO:l; protein, SEQ ID NO:2) by the use of oligos:
  • AT233 5'-CAAAGGAAGCTTTTTATTCAGGAGG-3' (SEQ ID NO:4); and AT240; 5'-CCAGGGATCCAATGGGGCCG-3' (SEQ ID NO:5).
  • PCRTM reactions were pooled, precipitated and resuspended in RO/DI water.
  • the PCRTM product was then blunt-end cloned into the commercial vector pBSIISK+ (Stratagene) that was opened at its EcoRV site.
  • This ligation product was transformed into XL-2 Blue competent cells (Stratagene) and colonies were screened by restriction enzyme digest. Multiple clones were derived and sequenced. Mutations were detected, compared with the Genbank reference sequences (SEQ ID NO:l and SEQ ID NO:2) and further sub-cloning was performed to generate a clone that encodes a completely wild-type protein.
  • One silent mutation introduced by the amplification process occu ⁇ ed in codon 246. This codon is changed from GTC to GTT. Each of these triplets code for alanine, so the LCAT protein sequence is unaffected.
  • a representative plasmid was named DN305 (nucleotide sequence is SEQ ID
  • the wild-type human LCAT in DN305 was sub-cloned into various mammalian expression vectors, starting with AC903.
  • AC903 contains a neomycin resistance gene driven by an SV40 promoter and a CMV promoter to drive the gene of interest.
  • This LCAT expression vector was named DO401.
  • Exemplary plasmids used in cell line development are shown in Table 15.
  • the DO401 and DW201 plasmids further comprise particular promoter structures.
  • the DO401 plasmid has the structure "CMV-TPL-intron-LCAT" and the DW201 plasmid has the structure "CMV-NP-TPL(full)-hexon-intron-LCAT".
  • the promoter is the basic CMV promoter;
  • the TPL is 35-173 of the 203 bp tripartite leader (TPL) sequence from the Ad5 adenovirus;
  • the intron is a synthetic intron (Kaufman and Sha ⁇ , 1982).
  • the promoter is still the basic CMV promoter; NP is the 5' untranslated region of the influenza virus nucleoprotein (NP) gene (Garfinkel and Katze, 1993); TPL is the full length (203 bp) tripartite leader sequence from the Ad5 adenovirus; the hexon is the 5' untranslated region of the Ad5 adenovirus hexon gene; and the intron is the same synthetic intron as in DO401.
  • NP influenza virus nucleoprotein
  • TPL is the full length (203 bp) tripartite leader sequence from the Ad5 adenovirus
  • the hexon is the 5' untranslated region of the Ad5 adenovirus hexon gene
  • the intron is the same synthetic intron as in DO401.
  • EXAMPLE 3 Secretory Cell Selection
  • the cells stably secrete therapeutic amounts of active LCAT and are amenable to further engineering, culturable and implantable.
  • the culturable and implantable requirements can be determined by growth and transgene expression in culture and survival and transgene expression in implantable devices, respectively.
  • the present example shows that cell lines derived from H04 cells have such advantages and are thus prefe ⁇ ed examples for use in the cell-based delivery of LCAT.
  • the following data show a comparison of different cell line production of recombinant secreted polypeptides, as exemplified human and rat growth hormone.
  • Separate cell lines derived from human ⁇ G H03 cells, human ⁇ G H04 cells and rodent (RIN) cells were expanded under standard tissue culture conditions to 80% confluency in a 25 cm tissue culture flask. Media was changed and 24 hr later a sample was taken for growth hormone measure. Cells were released by trypsinization, and cell number per flask was determined by hemocytometer count.
  • Human growth hormone concentration in the media was determined by ELISA assay, and rat growth hormone concentration was determined by semiquantitative western blot.
  • the output of secreted products from cell lines derived from human H03 and H04 cells and rat RIN 1046-38 cells is shown in Table 16.
  • H04-derived cells evidently had a higher output of both human and rat growth hormone than H03-derived cells. This indicates that H04-derived cells are the best protein factory.
  • the H04-derived human cell line output of secreted products also approached that of rodent-derived cells (RIN 1046-38) producing human and rat growth hormone.
  • the implantable devices were flexible rods obtained from Gore Hybrid Technologies, Inc. (GHT; Flagstaff, Arizona, U.S.A.), prepared as generally described in U.S. Patent No. 5,980,889, inco ⁇ orated herein by reference, and modified to include a wet seal component.
  • BG 636/17 (derived from BG H03) and BG 784/18 (derived from BG H04) cell lines were expanded using standard tissue culture conditions.
  • the cells were released from tissue culture flasks by trypsinization and loaded into implantation devices.
  • the devices were maintained in tissue culture. After 1 or 5 weeks, devices were removed, fixed with 10% neutral buffered formalin, paraffin embedded, and serially sectioned at a thickness of 50 ⁇ m.
  • Sections were stained with Hematoxylin and Eosin, and evaluated under a microscope.
  • the percentage of available space occupied in the device was essentially the same for the cell lines at 1 and 5 weeks.
  • the H04-derived cell line clearly filled more available space in the device than the H03 derived cell line.
  • the H04-derived cell line also out-performed HT-1080 cell lines in device occupancy studies, although the benefits were less marked than in comparison to H03 -derived cell lines.
  • H04 and H28 cell types were loaded into endoskeleton devices and cultured in tissue culture media for 5 weeks.
  • the H04 cells again demonstrated strong growth within the device, resulting in multiple sheets of cells.
  • H04 has essentially full occupancy for device loading and achieves, or at least approaches, 100% viability.
  • the H28 cells showed a decline in the number of cells with the expected cell debris form such cell death.
  • H04-derived cells clearly function significantly better in devices than either H03- or RiN-derived cells.
  • the dramatic improvement over RIN cell production is particularly impressive as rat growth hormone output from RIN cells in monolayer culture was as good or better than from H04 cells (see Table 16).
  • the effective device survival of H04 cells evidences high density growth and low nutrient requirements.
  • Cells of the H04, H28 and H03 cell types were loaded into endoskeleton devices and implanted subcutaneously in the backs of nude rats. Devices were left for 4 weeks, explanted, fixed, sectioned and histologically analyzed.
  • H04 cells grew as thick, densely packed masses and filled the void volume of the endoskeleton.
  • the H28 cells also grew at approximately the same rate to the same density.
  • H04 also has full occupancy in a device setting in vivo and again achieves, or at least approaches, 100% viability.
  • H03 cells When implanted subcutaneously in the backs of nude rats, H03 cells did not grow as well as H04 cells. This includes both the average number of troughs containing cells and the void volume occupancy by a viable cell mass. A necrotic core was observed in devices implanted with H03 cells, and a 30-50% loss of viability.
  • the superior cell line for pre-implantation culturing is H04, based on full viability and in contrast to the cell death seen with the H28 cell line.
  • the superior cell line for growth in vitro and in vivo is H04, considering the poor growth of H28 in vitro and the poor survival of H03 in vivo.
  • Example 3 shows that H04 cells have culturable and implantable properties that provide advantages for the cell-based delivery of LCAT.
  • the present example shows that H04 cells have further desirable properties, particularly that they express acceptable levels of LCAT and that they possess the post-translational processing machinery necessary to produce biologically active
  • H28 cell line yields few to no clones when transfected with the LCAT expression plasmid.
  • LCAT Activity In addition to the Western analysis, conditioned media samples were collected for LCAT enzyme analysis to confirm that the protein observed is LCAT and that it is a functional peptide. Certain of these assays were performed at the University of North Texas Health Science Center at Fort Worth, Forth Worth, TX.
  • the assay was performed by drying under nitrogen the following reagents: 260 ⁇ l of
  • EYPC egg yolk phosphatidylcholine
  • UC unesterified cholesterol
  • 3H-UC 3H-UC
  • This solution is rapidly injected by a 1ml syringe with a >25 gauge needle into 10ml assay buffer (lOmM TRIS pH 7.4, 5mM EDTA, 0.15M NaCl).
  • This solution is concentrated in an Amicon YM-30 membrane to a volume of no more than 2ml. The final volume is adjusted to 2.5ml with assay buffer.
  • a small test tube the following are added: 30 ⁇ l ethanolosome, 10 ⁇ l apoA-1, 60 ⁇ l BSA/2-mercaptoethanol, 50 ⁇ l of the sample to be tested and 50 ⁇ l assay buffer. This is incubated at 37°C for one hour. 3 ml propanol is then added to the tube to stop the reaction. The sample is centrifuged for 10 minutes at 3000 ⁇ m and the supernatant is transfe ⁇ ed to a new tube to evaporate. 30 ⁇ l chloroform is then added to each tube. This is then vortexed and spotted onto a TLC silica plate. The free cholesterol is then separated from the esterified cholesterol by capillary action and radioactive measurements are made of the separated samples.
  • LCAT human LCAT was produced in a number of cell lines derived from H04 cells.
  • the LCAT has a specific activity that at least approximates to normal, which co ⁇ elates with a normal glycosylation pattern.
  • the glycosylation structure and enzyme activity of recombinant LCAT from various sources has been analyzed in comparison to LCAT in human plasma (Miller et al, 1996).
  • the glycosylation pattern of LCAT prepared by baculoviral expression in insect (SF) cells was significantly different to normal (Miller et al., 1996). Perhaps su ⁇ risingly, HepG2 (liver) cells also produced LCAT with a different glycosylation pattern.
  • the insect cell and HepG2 cell forms of LCAT also had lower enzyme activity than normal (Miller et al, 1996).
  • the glycosylation pattern of LCAT expressed in CHO (Chinese hamster ovary) cells was found to be similar to that in plasma LCAT.
  • Mc-7777 cells also have specific activities lower than human plasma LCAT, despite the fact that Mc-7777 cells are hepatic cells (Ayyobi et al, 2000).
  • the recombinant LCAT from BHK and Mc-7777 cells have glycosylation patterns different to that of LCAT in human plasma, with that from BHK cells being significantly. This confirms the co ⁇ elation between glycosylation and LCAT activity in Miller et al. ( 1996).
  • the different glycosylation patterns of certain forms of recombinant LCAT correlate with their lower specific activities.
  • the human LCAT protein produced in H04 cells has a specific activity the same as, or at least approximating to, that of normal human LCAT from plasma, it can be assumed that H04 cells are producing LCAT with a normal or near- normal glycosylation pattern.
  • EXAMPLE 5 Expression of Therapeutic Levels of Human LCAT
  • H04 The advantageous culturable and implantable properties of H04 and the ability of these cells to produce properly processed, biologically active LCAT are shown in Example 3 and Example 4.
  • the present example further shows that H04 cells are capable of producing levels of LCAT within realistic therapeutic ranges.
  • Exemplary iterative engineering strategies involve the following steps. First and second, express a cell kill system (negative selectable marker) for safety reasons, and express human
  • Step 1 Express a kill system for safety reasons
  • Step 3 Express a second copy of LCAT for greater expression
  • Table 19 shows the LCAT secretion rates of various exemplary cell lines in plate culture, each containing only one copy of the LCAT transgene (1 hit).
  • One Unit (U) is 1 nmol of cholesterol esterified per hour (1 nmol cholesterol converted to cholesterol ester per hour).
  • Cell lines 1038/103 and 1069/111 have been grown and passaged repeatedly so that the population doubled a great number of times. These cell lines were then assayed against a low passage of the same cell line that had been preserved in the freezer. Table 20 shows the percent change in the output of the cell line over a given number of population doublings.
  • Implantable device designs were put into a variety of implantable device designs, all obtained from GHT.
  • Prefe ⁇ ed implantable devices are flexible rods prepared as generally described in U.S. Patent No. 5,980,889, inco ⁇ orated herein by reference, and modified to include a wet seal component. The devices tested were used to condition media in plate culture.
  • the 1069/111 cells express 24-164 units LCAT/million cells/day.
  • the relatively broad range of activities for this one cell line is due to the variety of device designs tested.
  • Samples were also drawn on a variety of days after implantation. The units of LCAT per million cells per day secreted after implantation in devices is lower than that observed in plate culture, but was expected (output in devices is typically lower than in culture even for cells that function relatively well in devices).
  • An LCAT-expressing H04-based cell line was also injected into a nude rat and a tumor allowed to grow. A higher serum level of LCAT was detected in comparison to control animals, showing that H04-dervied cells maintain function in vivo.
  • the present inventors do not consider that the foregoing high levels of LCAT are necessary to treat a deficient patient. In contrast, the inventors conclude that restoration of a patient to various sub-wild type levels will be beneficial in different ways.
  • a restoration to about 5% wild-type level forms the baseline of the present invention. Restoration to about 20% wild- type levels and about 50% wild-type levels may be prefe ⁇ ed in certain situations.
  • LCAT converts 1 nanomole of cholesterol to cholesterol ester in 1 hour (nmol/hr).
  • a defined number of cells 100,000 are cultured in a defined amount of media (3 ml) for a defined period of time (1 day).
  • the engineered cells such as H04 cells, secrete LCAT into the media.
  • the media is then assayed as described above (25 ⁇ l/reaction). With this information, the output of the cells can be defined as LCAT units secreted/million cells/day.
  • the dosage of cells needed to achieve a desired level of LCAT activity in a patient can then be readily calculated, as would be straightforward to those of ordinary skill in the art (e.g., see London et al, 1994; Treco et al, 1994).
  • the adjustments necessary for the decay in the total amount of LCAT over the course of one day mean that the actual plasma levels achieved would be slightly lower than those designed without accounting for the decay. However, as these factors are known, they can be readily accounted for in the initial considerations. For example, taking the foregoing 1069/111 cells, the calculations will be:
  • LCAT in plasma has a half-life of 4.5 days and, as it is secreted by the cells, will accumulate to the desired level (20 units/ml) over a short period of time.
  • the LCAT in the system equals the amount already in the system plus one day's output minus the decay of the total amount over the course of one day.
  • the formula for decay is:
  • h the half-life of the compound (LCAT's half-life in plasma is about 4.5 days)
  • t the number of days (in this case 1 day)
  • O d the daily output of a given cell line Assuming that the patient has no LCAT to start with, and that 13.2 million 1069/111 cells are provided, the formula means:
  • LCAT level of LCAT eventually levels out at 54069 units LCAT. In 3000 ml plasma, this gives 18.023 units/ml plasma. This is about 18% of normal wild-type levels.
  • Certain prefe ⁇ ed methods for cell implantation are those using rods and rod-like devices.
  • Rods are prefe ⁇ ed as the surface area to volume ratio facilitates proper functioning of the implanted cells.
  • the devices of U.S. Patent No. 5,980,889, preferably those modified to include a wet seal, may be formulated for use as rods and easily used to deliver 375 million cells or more.
  • rods are loaded with cells at a density of about a million cells per cm of device.
  • the rods are flexible before and after loading, so that the cell-loaded devices may be implanted within an animal or human subject in essentially any format.
  • a rod in linear form may be implanted within a limb.
  • the rods may be coiled to any desired shape and size.
  • Cu ⁇ ently prefe ⁇ ed formats include those wherein the cell-loaded rods are adapted to form a patch, as exemplified by patches of about 12 cm square, with a 2 mm thickness.
  • LCAT expression from engineered secretory cells can be readily used to provide therapeutically significant levels of LCAT upon implantation in vivo.
  • engineered secretory cells should be stable to bulk production in a bioreactor, harvesting, freezing and thawing. Cells undergoing this process should continue to secrete complex, fully biologically active polypeptides into the growth media with no significant differences in the response to secretagogues pre-bulk, post-bulk and post- thaw.
  • ⁇ G 49/206 cells were selected as a representative engineered ⁇ cell line to undergo the process of bulk production, harvest, freeze and thaw.
  • ⁇ G 49/206 is an engineered ⁇ cell line that reproducibly responds to a variety of secretagogues. This line has been engineered to stably express functional glucose transporter (GLUT-2) and glucokinase proteins and biologically active human insulin (Clark et al, 1997).
  • ⁇ G cell lines were bulk produced in the CellCubeTM system (Corning Costar) and frozen.
  • Frozen vials of cells representing each stage of the bulk production process were thawed and allowed to recover prior to testing their insulin response to various secretagogues.
  • the cells were ready to plate for testing of cell response to various secretagogues 48-72 hours after thawing.
  • Representative samples from each step were analyzed for response to various secretagogues.
  • the secretory response of pre-bulk, post bulk and harvest, and freeze/thaw samples was studied. The data show that there is no appreciable difference in the overall secretion response of cells that have undergone bulk production, bulk freezing and thawing.
  • the 96-well format has long been the standard used as it lends itself to automation and robotics handling.
  • handling attachment- dependent cell culture in the 96-well format needs to be conducted carefully when there is a need for several exchanges of solution.
  • the forces of surface tension associated with the meniscus on the well wall can stress and even damage cells on the bottom of the well as aqueous solutions are removed or added.
  • the shear forces created by a suction device e.g., a pipet tip
  • cells are left exposed directly to air. Any direct exposure to air is undesirable and causes stress to the cells; it may result in impaired and unpredictable function and response.
  • ⁇ G 49/206 cells were encapsulated in alginate using the following procedure. Trypsinized and PBS-washed cells are evenly suspended in a 1.5-2%) final concentration of sodium alginate (50:50 mixture of LV low viscosity and HV high viscosity, Kelco, CA) in growth medium without serum. The suspension is loaded in a syringe and then dispensed through a 27 gauge needle at approximately 0.3 ml/min. The droplets leaving the tip of the needle are blown off by a continuous air stream. By adjusting the velocity of the air stream, beads averaging approximately 800 ⁇ m can be achieved reproducibly.
  • the droplets are blown into a container holding a 1.35% (w/v) CaCl 2 /20 mM HEPES solution.
  • the beads are allowed to fully congeal for approximately 10 min in the CaCl 2 solution. Beads are washed twice in growth medium without serum and placed a T-flask with regular growth medium and incubated for about 72 hours with one feeding at 48 hours.
  • the beads were transfe ⁇ ed into a 50-ml conical and the total volume adjusted so the settled bead slurry makes up approximately 50% of the volume.
  • 50 ⁇ l bead slurry (about 30,000 cells) is dispensed into each well. Washing, stimulation, and assaying is performed as described above.
  • the encapsulated engineered cells were tested for responses to secretagogues relative to non-encapsulated cells.
  • the data generated shows that it is possible to encapsulate engineered cells and maintain comparable responses to secretagogues relative to non-encapsulated cells.
  • the responses in stimulation were essentially equivalent.
  • the data for encapsulated engineered cells also had a na ⁇ ow range of standard deviation values, indicative of better control of total remaining cell number at time of stimulation and of more stable conditions for all cells in the individual wells.
  • EXAMPLE 8 Maintenance of Secretion Performance in 96- Well Format
  • HTS high through put screening
  • Cells were plated, cultured, and assayed in 48-well dishes (100,000/well). For 96-well assays, ⁇ G 49/206 cells (30,000 per well) were plated and cultured for 48 hrs. in 150 ml of BetaGene Medium/ 2.5% fetal bovine serum; washed twice (20 min each, in 200 ml in HBSS), and stimulated with glucose or glucose plus IBMX.
  • the pattern of secretory responsiveness is maintained when ⁇ G 49/206 cells were plated, cultured, and assayed in a 96-well format: the inclusion of diazoxide in the medium provides a slight clamp to basal secretion, glucose alone is potently stimulatory, and the glucose response can be augmented by the inclusion of IBMX as a secretagogue.
  • the receptors include: ⁇ 2-adrenergic receptor (Genbank Ml 8415, M23533); glucagon-like peptide I receptor (Genbank L23503, U10037, U01156, U01104); somatostatin receptor V (Genbank AF004740, L14865, L14856, M81830, M96738, M81829, L07833); SUR channel (Genbank L78207, U63455, L78243); KIR channel (Genbank D50582); pancreatic polypeptide receptor (Genbank Z66526, U42387, U42389); muscarinic receptor (Genbank X52068, XI 5264, XI 5265, XI 5266, AF026263); glucocorticoid receptor (Genbank M10901, Ml 1050); GIP receptor (Genbank M10901, Ml 1050); GIP receptor (Genbank M10901, Ml 1050); GIP receptor (Genbank M10901, Ml
  • DNAs encoding the receptors were ligated into plasmids suitable for the stable transfection of mammalian cells.
  • Such plasmids contain genes that confer resistance to antibiotics and cloning sites for transgene insertion and expression.
  • Resistance to hygromycin hygromycin phosphotransferase
  • pCB7 Resistance to zeomycin is encoded in CW102 (pZeocmv).
  • CW102 was created by replacing the SV40 promoter in pZeoSV with the CMV promoter.
  • pZeoSV was digested with Bam HI and the ends were blunted-ended by a fill-in reaction with Klenow.
  • the CMV promoter was excised from pAC/CMV by digestion with Not I and prepared for blunt-end ligations by treatment with Klenow. There are two copies of the CMV promoter in CW102: one driving the expression of the zeomycin resistance gene and the other for transcribing transgenes of interest.
  • BetaGene Medium containing 7.8 mM glucose and supplemented with 3.5% fetal bovine serum (JRH Biosciences, Lenexa, KS), 100 milliunits/ml penicillin and 100 ⁇ g/ml streptomycin. Cells were passaged weekly using 0.05% trypsin-EDTA solution and cultured in an atmosphere of 95% air and 5% CO 2 at 37°C.
  • cell lines were grown to 50 to 75% confluence, harvested by trypsinization, washed once with phosphate-buffered saline (PBS), and resuspended in PBS for counting.
  • PBS phosphate-buffered saline
  • 1 x IO 7 cells were pelleted by centrifugation at 1000 rpm for 2 minutes and resuspended in 0.4 ml electroporation buffer (137 mM NaCl, 6 mM glucose, 5 mM KCl, 0.7 mM Na 2 HPO 4 , 20 mM Hepes, pH 7.0); or in BetaGene medium without serum. DNA was added to the cell suspension to achieve a final concentration of 30-50 ⁇ g/ml.
  • DNA was electroporated into the cells in a 2 mm cuvette at 170 volts, 510 ⁇ F and 129 ohms using an Electro Cell Manipulator 600 (BTX, Inc.). Stably transfected cells were selected by culturing in the appropriate drug for about 2 weeks.
  • the drug concentrations used were 500 ⁇ g/ml active fraction G418 (Geneticin, Gibco Life Sciences); 300 ⁇ g/ml for hygromycin (Boehringer Mannheim); 400 ⁇ g/ml for zeocin (Invitrogen).
  • the gene encoding the human ⁇ 2-adrenergic receptor ( ⁇ 2AR) inserted into a plasmid backbone was purchased from the American Type Culture Collection. Following replication and preparation of this plasmid, the DNA was designated BX700.
  • BX700 plasmid DNA was digested with restriction endonucleases to release the ⁇ 2AR genomic fragment. This fragment was ligated into pBluescript II SK plasmid (Stratagene), treated with the large fragment (Klenow) of DNA polymerase I to fill-in the overhangs created by digestion, and dephosphorylated with calf intestinal alkaline phosphatase (CIAP).
  • the plasmid resulting from this ligation, CE406, was digested, and the ⁇ 2AR DNA was ligated into pCB7 to create CE616 plasmid DNA.
  • RNAzol B RNA isolation reagent (Cinna/Biotex Laboratories International). RT-PCRTM was performed using the TitanTM One Tube RT-PCRTM System (Beohringer Mannheim).
  • RNA For the amplification of a portion of the rat GLP- 1 receptor mRNA, 100 ng of B 17/1 total RNA was transcribed at 55°C using AMV reverse transcriptase and amplified with a blend of Taq DNA polymerase and Pwo DNA polymerase. 35 rounds of amplification were performed with denaturation at 94°C (30 sees), annealing at 59°C (45 sees) and extension at 68°C (2 min.) using oligonucleotides IDK4 and IDK5.
  • the full- length human GLP-1 mRNA was amplified from RNA isolated from a human small cell lung line (ATCC: HTB-184, NCI: H510A) using oligonucleotides IDK3 and IDK6.
  • the rat GLP-1 cDNA was subcloned into pNOTA/T7 (5' to 3', Inc) to create plasmid CU201.
  • the human PCRTM product was subcloned into pBluescript KS that had been digested with EcoR V and the resulting plasmid was designated CX800.
  • the GLP-1 receptor fragment was isolated from CX800 following digestion and ligated with CW102 that had been digested.
  • the human pancreatic polypeptide receptor (PPR) mRNA was amplified from RNAs isolated from human lung cell lines (ATCC number CRL-5816; NCI-H810) using the TitanTM One Tube RT-PCRTM System. lOOng of total RNA was transcribed at 55°C; 35 rounds of amplification were performed with 94°C denaturation (30 sees), 57°C annealing (30 sees), and 68° C extension (2 min). PCRTM products were subcloned into pBluescript SK that had been digested with Hind III and filled in with Klenow to create plasmid DG105. The PPR fragment form DG105 was ligated into CW102 as a EcoR I/Kpn /fragment.
  • the mouse somatostatin receptor, type V gene ligated into pBluescript was obtained from
  • CWOOO was digested with PpuM I and treated with
  • the SSTRV DNA was ligated in CW102 that had been digested with Bam HI and treated with Klenow and CIAP, and the resulting plasmid was designated CX503.
  • Epinephrine participates in regulating circulating glucose levels by stimulating glucose production from the liver and inhibiting insulin secretion from the pancreatic ⁇ cell.
  • ⁇ G18/3El cells are relatively refractory to epinephrine and Clonidine, an analogue of epinephrine.
  • Clonidine an analogue of epinephrine.
  • human pancreatic islets are about 10-fold more sensitive to this compound than ⁇ G18/3El cells. It was reasoned that the sensitivity of ⁇ G18/3El cells to Clonidine could be increased by the transgenic overexpression of the ⁇ 2AR.
  • ⁇ G18/3El cells were electroporated (EP265) with plasmid CE616. Following selection with hygromycin and growth, single colonies were assayed by immunocytochemisty for the expression of the transgenic ⁇ 2AR.
  • ⁇ G18/3El cells and single clones derived from EP265 were plated on Falcon 8-chamber culture slides and maintained for 2 days in BetaGene Medium. Following fixation, cells were incubated with ⁇ 2AR antibody (diluted 1 :200; Dr. John Regan, University of Arizona, Arlington). Following incubation with a secondary antibody (antichicken IgG, alkaline phosphatase), immune complexes were detected colormetrically. The specificity of the ⁇ 2AR antibody was confirmed by competition assays with a ⁇ 2AR-glutathione-S transferase fusion protein.
  • one clonal cell line overexpressing ⁇ 2AR ( ⁇ G265/2), was about 10-fold more sensitive to Clonidine than human pancreatic islets and about 100-fold more sensitive than ⁇ G18/3El cells.
  • ⁇ G265/2 cell lines were encapsulated in alginate and injected into the intraperitoneal cavity of Zucker diabetic rats to show that the enhanced sensitivity to Clonidine extends to in vivo conditions. Beads were maintained in vivo for 3-5 days, or until blood glucose normalized. Animals were injected with Clonidine, an agonist of the ⁇ 2AR (50 ⁇ g/kg) or Yohimbine, an antagonist of the ⁇ 2AR (75 ⁇ g/kg). Blood glucose, rat C-peptide II, and human insulin levels were monitored at 20 minute intervals post-injection.
  • Clonidine injection resulted in a 50% reduction of human insulin in plasma
  • Somatostatin is a peptide hormone that has been shown to inhibit the release of growth hormone, thyroid stimulating hormone, insulin, and glucagon.
  • SS-28 and its analogue Octreotide may inhibit growth of some tumors.
  • Preliminary studies indicated that RIN 1046-38 clonal cell lines were insensitive to SS-28. Described here is the overexpression of mouse somatostatin receptor, type V gene (SSTRV) in a clonal derivative of RIN 1046-38 cell lines. An SSTRV-expressing cell line is analyzed with regard to the effects of SS-28 on insulin secretion.
  • SSTRV mouse somatostatin receptor, type V gene
  • the mouse somatostatin receptor, type V gene (SSTRV, Genbank accession number AF004740) ligated into pBluescript and a rabbit polyclonal antibody that recognizes the receptor (Ab9462) were received from the Dr. F. Charles Brunicardi, Baylor Medical Center, Houston, Texas. Following replication of the plasmid, the DNA was designated CWOOO. CWOOO was digested with PpmM I and treated with Klenow. The SSTRV DNA was ligated in CW102 that had been digested with BCW000 was digested with PpmM I and treated with Klenow. The SSTRV DNA was ligated in CW102 that had been digested with Bam HI, filled in with Klenow, and treated with CIAP, and the resulting plasmid was designated CX503.
  • SSTRV Genbank accession number AF004740
  • Ab9462 rabbit polyclonal antibody that recognizes the receptor
  • ⁇ G 40/110 cells (clonal derivatives of RIN 1046-38 overexpressing human insulin and glucokinase) were transfected (EP 603) with plasmid CX503. Following selection in Zeomycin, 13 colonies were selected for further analysis and growth. Portions of the clones were plated onto cover slides and assayed by immunocytochemistry for the expression of SSTRV. The primary antibody Ab9462 was diluted 1/1000 and immune complexes were colorimetrically detected following incubation with a secondary antibody, goat anti-rabbit linked alkaline phosphatase. Of the 13 clones, one was a high expressor of SSTRV ⁇ G 603/11), and two expressed low levels of the receptor ( ⁇ G 603/8 and 10).
  • SS-28 was tested as an inhibitor of various secretagogues of insulin secretion. As shown herein, at 5 nM SS-28 effectively inhibits stimulated insulin secretion in the presence of BetaGene Medium with no glucose and under conditions of maximum stimulation, Stimulatory Cocktail (BetaGene Media supplemented with 10 mM glucose, 10 mM each of glutamine, leucine, and arginine, 100 ⁇ M carbachol, and 100 ⁇ M IBMX). The expression of the somatostatin receptor, type V gene (SSTRV) thus successfully inhibits the secretory responses.
  • Stimulatory Cocktail BetaGene Media supplemented with 10 mM glucose, 10 mM each of glutamine, leucine, and arginine, 100 ⁇ M carbachol, and 100 ⁇ M IBMX.
  • SSTRV somatostatin receptor, type V gene
  • EXAMPLE 12 Expression of Negative Selectable Marker for Targeted Cell Killing
  • the present example describes the successful installation of a safety mechanism to allow cells to be killed after implantation, e.g., in scenarios such as malfunction, host incompatibility and/or breach in an encapsulation device.
  • the cells are provided with a negatively selectable marker or kill system in the form of the GLUT-2 glucose transporter, which may be used in combination with the toxin, streptozotocin (STZ).
  • ⁇ G 498/20 cells a human neuroendocrine cell line engineered to express insulin, was tested for sensitivity to STZ and found to be resistant to cell killing at concentrations up to 10 mM.
  • ⁇ G 498/20 cells were electroporated (EP642) with plasmid AD402 (CMVp- GLUT2/SV40p-Hygro), selected for resistance to hygromycin, and tested by Western blotting for the expression of the GLUT-2 transporter.
  • ⁇ G 642 clones expressed variable levels of the transgenic GLUT-2, and those cells transfected with a plasmid confe ⁇ ing hygromycin resistance alone ( ⁇ G 640-v) did not express detectable levels of the transporter.
  • High GLUT-2 expressors were most sensitive to STZ, with certain cell lines effectively killed at less than 3 mM. These data prove the feasibility of converting a human cell line from one that is STZ-resistant to a STZ-sensitive phenotype by the overexpression of the GLUT-2 transporter, thus providing the required safety mechanism for in vivo implantation.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefe ⁇ ed embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • LCAT lecithin-cholesterol acyltransferase
  • Albers et al Familial lecithin-cholesterol acyltransferase deficiency in a Japanese family: Evidence for functionally defective enzyme in homozygotes and obligate heterozygotes, Hum. Genet, 62:82, 1982.
  • Albers et al Defective enzyme causes lecithin-cholesterol acyltransferase deficiency in a Japanese kindred, Biochim. Biophys. Acta., 835:253, 1985.
  • Clark et al Endocrinology, 127:2779-2788, 1990. Clark et al, Human Gene Therapy, 6:1329-1341, 1995.
  • Graham and Prevec Biotechnology, 20:363-390, 1992. Graham and Prevec, In: E.J. Mu ⁇ ay (ed.), Methods in Molecular Biology: Gene Transfer and Expression Protocol, Clifton, NJ: Humana Press, 7:109-128, 1991. Graham and van der Eb, Virology, 52:456-467, 1973.
  • JAMA, 1984b The lipid research clinics coronary primary prevention trial results, I: reduction in incidence of coronary heart disease, JAMA, 251:351-364, 1984a.
  • JAMA, 1984b The lipid research clinics coronary primary prevention trial results, II: the relationship of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA, 251 :351-364, 1984b.
  • Kaneda et al Science, 243:375-378, 1989. Kaplitt et al, Nature Genetics, 8:148-154, 1994.
  • Nicolas and Rubinstein In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, pp. 494-513, 1988.
  • Tur-Kaspa et al Mol. Cell Biol, 6:116-718, 1986. Usdin et al, Endocrinology, 133:2861-2870, 1993. WagnCT ⁇ f ⁇ /., Science, 260: 1510-1513, 1990. Walsh et al, Proc. Natl Acad. Sci. USA, 89:7257-7261, 1994. Warnock and Rajotte, Diabetes, 37:467-470, 1988. Wong et al., Gene, 10:87-94, 1980.

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Abstract

L'invention concerne des compositions, des kits et des dispositifs comprenant des cellules obtenues par génie génétique sécrétant des niveaux thérapeutiquement efficaces de lécithine cholestérol acyltransférase (LCAT). L'invention concerne également des méthodes d'utilisation desdites cellules dans le cadre de diverses applications diagnostiques et thérapeutiques, notamment pour le traitement efficace des états liés à une déficience en LCAT, tels que l'athérosclérose, et ce au moyen de doses de cellules étonnamment faibles et sur la base d'analyses pronostiques.
PCT/US2000/019047 1999-07-14 2000-07-13 Compositions a base de lignees cellulaires de recombinaison secretant lcat, et methodes associees WO2001005943A2 (fr)

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AU59316/00A AU5931600A (en) 1999-07-14 2000-07-13 Lcat recombinant cell line compositions and methods

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WO2010144904A1 (fr) * 2009-06-12 2010-12-16 Alphacore Pharma Llc Utilisation de lcat pour traiter l'anémie et le dysfonctionnement des cellules de globules rouges
US8168416B2 (en) 2007-07-26 2012-05-01 Amgen Inc. Modified lecithin-cholesterol acyltransferase enzymes

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Cited By (9)

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Publication number Priority date Publication date Assignee Title
US8168416B2 (en) 2007-07-26 2012-05-01 Amgen Inc. Modified lecithin-cholesterol acyltransferase enzymes
US8703926B1 (en) 2007-07-26 2014-04-22 Amgen Inc. Modified lecithin-cholesterol acyltransferase enzymes
US9006408B2 (en) 2007-07-26 2015-04-14 Amgen Inc. Modified lecithin-cholesterol acyltransferase enzymes
WO2010144904A1 (fr) * 2009-06-12 2010-12-16 Alphacore Pharma Llc Utilisation de lcat pour traiter l'anémie et le dysfonctionnement des cellules de globules rouges
CN102802659A (zh) * 2009-06-12 2012-11-28 阿尔法科制药有限责任公司 Lcat治疗贫血和红细胞功能障碍的用途
US8492108B2 (en) 2009-06-12 2013-07-23 Bruce J. Auerbach Methods of treating anemia and red blood cell dysfunction with lecithin cholesterol acyltransferase
AU2010259875B2 (en) * 2009-06-12 2015-10-22 Alphacore Pharma Llc Use of LCAT for treating anemia and red blood cell dysfunction
CN102802659B (zh) * 2009-06-12 2015-11-25 阿尔法科制药有限责任公司 Lcat治疗贫血和红细胞功能障碍的用途
RU2576838C2 (ru) * 2009-06-12 2016-03-10 МЕДИММЬЮН, ЭлЭлСи Использование лхат для лечения анемии и дисфункции красных кровяных клеток

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