WO1997014441A1 - Method for restoring glucose responsiveness to insulin secretion - Google Patents
Method for restoring glucose responsiveness to insulin secretion Download PDFInfo
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- WO1997014441A1 WO1997014441A1 PCT/US1996/016736 US9616736W WO9714441A1 WO 1997014441 A1 WO1997014441 A1 WO 1997014441A1 US 9616736 W US9616736 W US 9616736W WO 9714441 A1 WO9714441 A1 WO 9714441A1
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- calbindin
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
Definitions
- the present invention relates generally to the field of genetic engineering and gene expression. More specifically, it relates to artificial tissues, particularly to artificial tissues comprising engineered cells having the ability to secrete insulin in a glucose-sensitive fashion. It also relates to the use of engineered cells in the production of human insulin for use in, for example, the treatment of insulin deficient conditions such as type I diabetes mellitus.
- the ⁇ cells of the pancreatic islets of Langerhans synthesize insulin. A portion of this insulin is slowly and continually released into the bloodstream (basal secretion). However, the bulk of the insulin is stored in secretory vesicles and released only in response to certain physiological stimuli (stimulated secretion).
- the principal physiological stimulus for the secretion of insulin is increased blood levels of glucose (e.g. , following ingestion of a carbohydrate meal).
- glucose e.g. , following ingestion of a carbohydrate meal.
- the capacity of normal islet ⁇ cells to sense a rise in blood glucose concentration and to respond by secreting insulin is critical to control of blood glucose levels.
- pancreas loses its ability to manufacture and secrete insulin in response to rising blood glucose concentrations.
- the result is a metabolic imbalance, which causes blindness, kidney-related diseases, neurological disorders, cardiovascular diseases, non-accidental amputation of limbs, and death.
- the current preferred treatment for insulin deficiency is injection of insulin once or twice daily.
- the objective of this regimen is to maintain glucose levels close to normal.
- daily insulin injections can not reproduce the rapid insulin secretory responses of normal islets to physiological demand.
- Most insulin deficient patients never achieve the finely-tuned glucose homeostasis needed to avoid long-term complications.
- the present invention comprises an engineered cell that secretes insulin in a glucose-sensitive fashion and that further expresses a drug sensitivity.
- This engineered cell expresses a first exogenous nucleic acid that encodes a calbindin molecule and a second exogenous nucleic acid that encodes a drug sensitivity, with the proviso that in the absence of said first exogenous nucleic acid that encodes a calbindin molecule, the host cell does not secrete insulin in glucose-sensitive fashion.
- the engineered cell may be a primary isolate, a continuous non-transformed cell line, or an insulinoma, preferably of human origin.
- the engineered cell may optionally be negatively selected by exposure to metabolites or drugs that are lethal to cells that express the drug sensitivity gene.
- the invention further encompasses a method for imparting glucose-sensitive insulin secretion to a host cell that does not exhibit glucose-sensitive insulin secretion, comprising the steps of a) providing a cell that is capable of producing and secreting insulin, but has an impaired ability to secrete insulin in a glucose sensitive fashion; and b) stably introducing into said cell a nucleic acid that encodes a calbindin molecule, wherein the expression of said nucleic acid that encodes a calbindin molecule imparts to the engineered host cell the ability to secrete insulin in a glucose-sensitive fashion.
- the mammal is a human patient
- the engineered cell is either con-specific or syngeneic with the mammal into which it is stably introduced, and the cell is selected from the group consisting of a primary isolate, a continuous non-transformed cell line or an insulinoma.
- inventions of this invention are artificial implantable tissues that secrete insulin in response to humoral signals, in particular glucose.
- One such embodiment is an engineered cell that expresses calbindin and secretes insulin in a glucose-sensitive fashion, wherein the cell is enclosed in a semipermeable matrix or membrane that permits the diffusion of glucose and nutrients into the matrix, and the diffusion of insulin and waste products out of the matrix.
- the matrix may be formed from plasma, fibrinogen, casein, fibrin, limulus lysate, milk protein, collagen, agarose, carrageenan, agar, alginate, guar gum, gum arabic, pectin, tragacanth gum, xanthan gum, and mixtures thereof.
- These matrix-enclosed engineered glucose-sensitive insulin-secreting calbindin- expressing cells are implanted or injected into an animal that is unable to secrete insulin in a glucose-sensitive fashion.
- a cell-containing matrix of this invention is prepared by: ( 1 ) polymerizing matrix components or precursors in the presence of viable engineered cells that express an exogenous calbindin and secrete insulin in a glucose-sensitive fashion, and (2) recovering a porous matrix containing viable cells.
- the polymerization can be achieved by exposing the respective precursor to a polymerization promoting reagent and/or a polymerization promoting condition.
- the current invention provides methods for growing artificial ⁇ cells in liquid culture and for the increased production of human insulin by perfusion of such recombinant cells with glucose-containing buffers.
- FIG. 1 Western blot analysis of cells that express calbindin
- FIG. 2 Northern blot analyses of calbindin-D 28k and insulin mRNA in RIN cells transformed with pREP-calbindin-D 28 .
- FIG . 3 Changes in intracellular calcium in response to calcium ionophore A231 87.
- the presently-available implantable artificial pancreatic devices suffer from problems of donor availability, tissue typing, and recovery and replacement of cells. These problems are addressed herein by the provision of renewable stocks of tissue culture cells that secrete insulin in a glucose- sensitive fashion.
- a cell secretes insulin at a rate and to an extent that exceeds basal secretion.
- basal secretion essentially the only insulin secreted is basal secretion.
- Glucose-sensitive stimulated secretion is triggered by glucose concentrations in the above mentioned range, and ceases when the glucose concentrations fall below this range.
- impaired ability to secrete insulin in a glucose-sensitive fashion refers to a cell or organism that cannot secrete insulin (i.e., because the ⁇ cells are absent or dysfunctional), or that cannot secrete insulin in a glucose-sensitive fashion.
- nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless specifically limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly provides the complementary sequence thereof, as well as the sequence explicitly indicated.
- the terms “nucleic acid” and “gene” are used interchangeably and they encompass "cDNA.
- exogenous nucleic acid generally denotes a nucleic acid that has been isolated, cloned and introduced into or expressed in a cell or cellular environment other than the cell or cellular environment in which said nucleic acid or protein may be found in nature.
- the term encompasses both nucleic acids originally obtained from a different organism or cell type than the cell type in which it is expressed, and also nucleic acids that are obtained from the same cell line as the cell line in which it is expressed.
- expression or “expressed” refers to transcription of nucleic acids, either without or with subsequent translation.
- nucleic acid sequence encoding refers to a nucleic acid which contains sequence information that, if translated, yields the primary amino acid sequence of a specific protein or peptide. This phrase specifically encompasses degenerate codons (i.e. , different codons which encode a single amino acid) of the native sequence or sequences which may be introduced to c ⁇ nform with codon preference in a specific host cell.
- calbindin denotes a family of genes and gene products which specifically encompasses the gene and gene product of the calbindin-D 28k (CaBP 28k ) or calbindin-D9K cDNA and conservatively modified variants thereof. Calbindin further denotes nucleic acid sequences and their gene products wherein the gene products bind calcium and are recognized by antibodies that specifically bind to the gene product of CaBP 28k cDNA.
- CaBP 28k calbindin-D 28k
- Calbindin-D9K cDNA calbindin-D9K cDNA and conservatively modified variants thereof.
- Calbindin further denotes nucleic acid sequences and their gene products wherein the gene products bind calcium and are recognized by antibodies that specifically bind to the gene product of CaBP 28k cDNA.
- Vitamin D-dependent calcium binding proteins chemistry, distribution, functional considerations, and molecular biology.: update 1 995.. in D.D. Bikle, ed. HORMONAL REGULATION OF BONE AND MINERAL METABOLISM. V4, p84-1 07 ( 1 995) .
- the sequences for the various calbindins have been reported in the literature, and are hereby incorporated by reference.
- the sequences of two calbindins, rat D28 and human D27, are included as sequence ID No 1 and
- the phrase "expresses a drug sensitivity" signifies 1 ) that a cell is rendered susceptible to one or more specific chemical substances by virtue of expressing a gene product that is capable of producing a cytotoxic chemical substance, or 2) that a cell that contains an inducible gene (i.e., placed under the control of an inducible promoter) whose gene product is itself cytotoxic.
- cytosine deaminase converts 5-fluorocytosine ("5FC") into the lethal metabolite 5-fluorouracil (" 5FU").
- 5FC 5-fluorocytosine
- 5FU 5-fluorouracil
- Another gene that confers drug sensitivity upon a host cell is the thymidine kinase gene, which confers sensitivity to gancyclovir. See: Ido et al. ( 1 995), Cancer Research, 55: 31 05; Izquierdo et al.
- con-specific indicates that two cells or cell lines or a cell line and an organism are from the same animal species.
- sergeneic means that two cells or a cell and an organism are from the same individual.
- nucleic acid or a protein when used with reference to a nucleic acid or a protein generally denotes that the composition or primary sequence of said nucleic acid or protein has been altered from the naturally occurring sequence using experimental manipulations well known to those skilled in the art. It may also denote that a nucleic acid or protein has been isolated and cloned into a vector, or a nucleic acid that has been introduced into or expressed in a cell or cellular environment other than the cell or cellular environment in which said nucleic acid or protein may be found in nature.
- Recombinant or “engineered” when used with reference to a cell indicates that the cell replicates or expresses a nucleic acid or expresses a peptide or protein encoded by a nucleic acid, whose origin is exogenous to the cell.
- Recombinant cells can express nucleic acids that are not found within the native (non-recombinant) form of the cell.
- Recombinant cells can also express nucleic acids found in the native form of the cell wherein the nucleic acids are re-introduced into the cell by artificial means.
- vector denotes an engineered nucleic acid construct that contains sequence elements that mediate the replication of the vector sequence and/or the expression of coding sequences present on the vector.
- examples of vectors include eukaryotic and prokaryotic plasmids, viruses, cosmids, phagemids, and the like.
- operably linked refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
- a nucleic acid expression control sequence such as a promoter, or array of transcription factor binding sites
- One or more selected isolated nucleic acids may be operably linked to a vector by methods known in the art.
- Engineerered or “transduction” or “transfection” denotes a group of processes whereby an exogenous nucleic acid is introduced into a cell, such that the cell is capable of replicating and/or expressing the exogenous nucleic acid.
- a selected nucleic acid is first inserted into a vector and the vector is then introduced into the cell.
- plasmid DNA that is introduced under appropriate environmental conditions may undergo replication in the engineered cell, and the replicated copies are distributed to progeny cells when cell division occurs. As a result, a new cell line is established, containing the plasmid and carrying the genetic determinants thereof.
- Transduction by a plasmid in this manner occurs at high frequency when the transducting plasmid DNA is in closed loop form, and does not or rarely occurs if linear plasmid DNA is used.
- Stably expressed indicates that the exogenous nucleic acid is expressed in a host cell essentially for the life of the cell, and that the progeny of the engineered cell, if any, also express the exogenous nucleic. Stable expression may be the consequence of integration of the nucleic acid into the host cell genome. Alternatively, the vector that comprises the nucleic acid may be replicated and transmitted episomally.
- “Stably introducing" a cell or cell line into an organism means that the cell or cell line is injected or implanted into the organism, for example intraperitoneally, into the blood, or into vascularized tissue, and that the cell or cell line is able to survive and perform for extended periods of time, measured in weeks, preferably months, and most preferably years.
- An important aspect of this invention are methods for introducing calbindin into insulin-producing cells.
- Standard eukaryotic transduction methods are used to produce cell lines which express calbindin and, optionally, a drug resistance gene. It is expected that those of skill in the art are knowledgeable in the numerous systems available for cloning and expressing nucleic acids.
- nucleic acid of interest e.g., one encoding calbindin
- promoter which is either constitutive or inducible
- the vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both.
- Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid.
- the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g.
- the expression vector typically comprises a eukaryotic transcription unit or expression cassette that contains all the elements required for the expression of exogenous calbindin in eukaryotic cells.
- a typical expression cassette contains a promoter operably linked to the DNA sequence encoding a calbindin protein and signals required for efficient polyadenylation of the transcript.
- Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
- the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
- the other upstream promoter elements determine the rate at which transcription is initiated.
- Enhancer elements can stimulate transcription up to 1 ,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types.
- enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus, the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
- the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
- the expression cassette should also contain a transcription termination region downstream of the calbindin structural gene to provide for efficient termination.
- the termination region may be obtained from the same source as the promoter sequence or may be obtained from a different source.
- polyadenylation sequences are also commonly added to the vector construct. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 1 1 -30 nucleotides upstream. Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40, or a partial genomic copy of a gene already resident on the expression vector.
- the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the transduced DNA.
- a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
- the vector may or may not comprise a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the transduced DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
- the vectors usually comprise selectable markers which result in nucleic acid amplification such as the sodium, potassium ATPase, thymidine kinase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD (carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase), adenosine deaminase, dihydrofolate reductase, and asparagine synthetase and ouabain selection.
- selectable markers which result in nucleic acid amplification such as the sodium, potassium ATPase, thymidine kinase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD
- high yield expression systems not involving nucleic acid amplification are also suitable, such as using a bacculovirus vector in insect cells, with calbindin encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
- the expression vectors of the present invention will typically contain both prokaryotic sequences that facilitate the cloning of the vector in bacteria as well as one or more eukaryotic transcription units that are expressed only in eukaryotic cells, such as mammalian cells.
- the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells.
- nucleic acid Once a nucleic acid is synthesized or isolated and inserted into a vector and cloned, one may express the nucleic acid in a variety of recombinantly engineered cells known to those of skill in the art. Expression of a an exogenous nucleic acid can be enhanced by including multiple copies of, for example, a calbindin-encoding nucleic acid in an engineered cell, by selecting a vector known to reproduce in the host, thereby producing large quantities of protein from exogenous inserted DNA (such as pUC8, ptac 1 2, or plN-lll-ompA1 , 2, or 3), or by any other known means of enhancing peptide expression.
- exogenous inserted DNA such as pUC8, ptac 1 2, or plN-lll-ompA1 , 2, or 3
- Calbindin molecules will be expressed when the DNA sequence is functionally inserted into a vector. "Functionally inserted” means that it is inserted in proper reading frame and orientation and operably linked to proper regulatory elements. Typically, a calbindin gene will be inserted downstream from a promoter and will be followed by a stop codon, although production as a hybrid protein followed by cleavage may be used, if desired.
- Vectors to which calbindin-encoding nucleic acids are operably linked may be used to introduce these nucleic acids into host cells and mediate their replication and/or expression.
- “Cloning vectors” are useful for replicating and amplifying the foreign nucleic acids and obtaining clones of specific foreign nucleic acid-containing vectors.
- “Expression vectors” mediate the expression of the foreign nucleic acid.
- the particular eukaryotic expression vector used to transport calbindin or any other gene into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic cells may be used. Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are typically used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV- 1 MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
- exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
- viral vectors such as retroviral vectors are useful for modifying eukaryotic cells because of the high efficiency with which the retroviral vectors transfect target cells and integrate into the target cell genome. Additionally, the retroviruses harboring the retroviral vector are capable of infecting cells from a wide variety of tissues.
- Retroviral vectors are produced by genetically manipulating retroviruses.
- Retroviruses are RNA viruses because the viral genome is RNA. Upon infection, this genomic RNA is reverse transcribed into a DNA copy which is integrated into the chromosomal DNA of transfected cells with a high degree of stability and efficiency. The integrated DNA copy is referred to as a provirus and is inherited by daughter cells as is any other gene.
- the wild type retroviral genome and the proviral DNA have three genes: the gag, the pol and the env genes, which are flanked by two long terminal repeat (LTR) sequences.
- the gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins.
- the 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAs. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsulation of viral RNA into particles (the Psi site). See Mulligan, R.C. ( 1 983), In: Experimental Manipulation of Gene Expression, M. Inouye (ed), 1 55- 1 73; Mann et al. ( 1 983), Cell, 33: 1 53- 1 59; Cone, R.D. and R.C. Mulligan ( 1 984), Proceedings of the National Academy of Sciences, U.S.A. , 81 : 6349-6353.
- retroviral vectors The design of retroviral vectors is well known to one of skill in the art. See Singer, M. and Berg, P., supra. In brief, if the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis acting defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Retroviral genomes from which these sequences have been deleted, as well as cell lines containing the mutant genome stably integrated into the chromosome are well known in the art and are used to construct retroviral vectors.
- the retroviral vector particles are prepared by recombinantly inserting a nucleic acid encoding a calbindin molecule and optionally a drug sensitivity gene into a retrovirus vector and packaging the vector with retroviral capsid proteins by use of a packaging cell line.
- the resultant retroviral vector particle is generally incapable of replication in the host cell and is capable of integrating into the host cell genome as a proviral sequence containing the calbindin nucleic acid.
- the patient is capable of producing calbindin and optionally the gene product of the drug sensitivity gene.
- Packaging cell lines are generally used to prepare the retroviral vector particles.
- a packaging cell line is a genetically constructed mammalian tissue culture cell line that produces the necessary viral structural proteins required for packaging, but which is incapable of producing infectious virions.
- Retroviral vectors lack the structural genes but have the nucleic acid sequences necessary for packaging.
- To prepare a packaging cell line an infectious clone of a desired retrovirus, in which the packaging site has been deleted, is constructed. Cells comprising this construct will express all structural proteins but the introduced DNA will be incapable of being packaged.
- packaging cell lines can be produced by transducing a cell line with one or more expression plasmids encoding the appropriate core and envelope proteins. In these cells, the gag, pol, and env genes can be derived from the same or different retroviruses.
- a number of packaging cell lines suitable for the present invention are available in the prior art. Examples of these cell lines include Crip, GPE86, PA31 7 and PG 1 3. See Miller et al. ( 1 991 ), J. Virol. , 65: 2220-2224, which is incorporated herein by reference. Examples of other packaging cell lines are described in Cone, R. and Mulligan, R.C. ( 1 984), Proceedings of the National Academy of Sciences, U. S.A. , 81 : 6349-6353 and in Danos, O. and R.C. Mulligan ( 1 988), Proceedings of the National Academy of Sciences, U. S.A. , 85: 6460-6464, Eglitis et al.
- cells may be transfected with adeno-associated viral vectors. See, e.g. , Methods in
- Adeno associated viruses require helper viruses such as adenovirus or herpes virus to achieve productive infection. In the absence of helper virus functions, AAV integrates (site-specifically) into a host cell's genome, but the integrated AAV genome has no pathogenic effect.
- the integration step allows the AAV genome to remain genetically intact until the host is exposed to the appropriate environmental conditions (e.g., a lytic helper virus), whereupon it re-enters the lytic life-cycle.
- a lytic helper virus e.g., Samulski (1 993), Current Opinion in Genetic and Development, 3: 74-80, and the references cited therein provides an overview of the AAV life cycle. See also West et al. ( 1 987), Virology, 1 60: 38-47; Carter et al. ( 1 989), U.S. Patent No. 4,797,368; Carter et al.
- Plasmids designed for producing recombinant vaccinia such as pGS62, (Langford et al. ( 1 986), Mol. Cell. Biol. , 6: 31 91 -31 99) may also be used.
- This plasmid consists of a cloning site for insertion of foreign nucleic acids, the P7.5 promoter of vaccinia to direct synthesis of the inserted nucleic acid, and the vaccinia TK gene flanking both ends of the foreign nucleic acid.
- pancreatic ⁇ cells A number of cell lines derived from pancreatic ⁇ cells, commonly known as insulinoma cells, are useful for the practice of this invention and are readily available.
- hamster insulinoma (HIT-T1 5) cells are well studied and are readily available from the American Type Tissue Collection.
- a number of rat insulinoma cell lines are also available.
- the RINm ⁇ F and RINrl 046-38 cell lines were derived from a radiation induced tumor of the islet beta -cells (Gazdar et al. , 1 980; Clark et al. , 1 990).
- MSL-G2 cells were derived from a liver metastasis of an islet cell tumor. These cells require periodic passage in an animal host in order to maintain expression of their endogenous insulin gene
- Kisanuki et al. ( 1 995), Expression of insulin receptor on clonal pancreatic alpha cells and its possible role for insulin-stimulated negative regulation of glucagon secretion, Diabetologia 38(4) : 422-9; Mogami et al. ( 1 994), Inhibition of ATP- sensitive K + channel by a non-sulfonylurea compound KAD-1 229 in a pancreatic beta-cell line, MIN 6 cell, Eur. J. Pharmacol. 269(3) : 293-8; Flatt et al. ( 1 990), Stimulaory effects of glucagon-like peptides on human insulinoma cells and insulin-releasing clonal RINm ⁇ F cells, Diabetes Res. 1 3(2) : 55-9.
- this invention also encompasses cells that express an exogenous insulin gene in addition to an exogenous calbindin gene.
- Such cells may be generated as described herein and by methods known in the art, using vectors that contain operably linked calbindin and insulin genes.
- Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transduction, polybrene, protoplast fusion, electroporation, liposomes, DEAE dextran, receptor-mediated endocytosis, micro-injection of the DNA directly into the cells, plasmid vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign nucleic acidic material into a host cell (see Sambrook er al. , supra) . It is only necessary that the particular genetic engineering procedure utilized be capable of successfully introducing at least one nucleic acid into the host cell which is capable of expressing the calbindin.
- Freshney 1 994) CULTURE OF ANIMAL CELLS, A MANUAL OF BASIC TECHNIQUE, third edition, Wiley-Liss, New York, Kuchler et al. ( 1 977), BIOCHEMICAL METHODS IN CELL CULTURE AND VIROLOGY, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc., and the references cited therein provide a general guide to the culture of cells. Cultured cell systems often will be in the form of monolayers of cells, although cell suspensions are also used.
- nucleic acid construct that encodes a calbindin and optionally a drug sensitivity gene
- Nucleic acids and proteins are detected and quantified herein by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC) , hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
- the detection of nucleic acids proceeds by well known methods such as Southern analysis, northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography.
- a variety of methods of specific DNA and RNA measurements and nucleic acid hybridization techniques known to those of skill in the art are useful for detecting and quantifying the presence and expression of calbindin, drug resistance genes, or insulin.
- one method for evaluating the presence or and levels of calbindin DNA and calbindin expression in a sample involves a Southern transfer. Southern et al. ( 1 975), J. Mol. Biol. , 98: 503. Briefly, the digested genomic DNA is run on agarose slab gels in buffer and transferred to membranes. Hybridization is carried out using probes that recognize a calbindin or an insulin sequence.
- a Northern transfer may be used for the detection of calbindin or insulin mRNA in samples of RNA from engineered insulin-producing cells that express the calbindin gene.
- the mRNA is isolated from a given cell sample using an acid guanidinium-phenol-chloroform extraction method. The mRNA is then electrophoresed to separate the mRNA species and the mRNA is transferred from the gel to a nitrocellulose membrane.
- labeled probes are used to identify the presence or absence of a calbindin or an insulin transcript.
- nucleic acid hybridization format is not critical.
- a variety of nucleic acid hybridization formats are known to those skilled in the art.
- common formats include sandwich assays and competition or displacement assays.
- Hybridization techniques are generally described in " Nucleic Acid Hybridization, A Practical Approach, " Ed. Hames, B.D. and Higgins, S.J., IRL Press, 1 985.
- the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected.
- a nucleic acid amplification system which multiplies the target nucleic acid being detected.
- In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q ?-replicase amplification and other RNA polymerase mediated techniques (e.g. , NASBA) are found in Berger, Sambrook, and Ausubel, as well as Mullis et al. ( 1 987), U.S. Patent No.
- Beta Replicase systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a select sequence is present.
- the select sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
- Oligonucleotides for use as probes e.g., in in vitro amplification methods, for use as gene probes, or as inhibitor components are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts.,
- oligonucleotides where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983), J. Chrom., 255: 137-149.
- the sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65: 499-560.
- In situ hybridization assays are well known and are generally described in Angerer et al. (1987), Methods Enzymol., 152: 649-660.
- in situ hybridization assay cells are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of calbindin-specific probes that are labelled.
- the probes are preferably labelled with radioisotopes or fluorescent reporters.
- calbindin or insulin may be detected or quantified by a variety of methods. Preferred methods involve the use of specific antibodies.
- IMMUNOLOGY Wiley/Greene, NY; and Harlow and Lane ( 1 989), ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Press, NY; Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding ( 1 986), MONOCLONAL ANTIBODIES : PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, NY; and Kohler and Milstein ( 1 975), Nature, 256: 495-497.
- Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1 989), Science, 246: 1 275- 1 281 ; and Ward et al. ( 1 989), Nature, 341 : 544-546.
- Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of at least about .1 mM, more usually at least about 1 ⁇ M, preferably at least about .1 ⁇ M or better, and most typically and preferably, .01 ⁇ M or better.
- the presence of a calbindin or insulin polypeptide (including peptide, transcript, or enzymatic digestion product) in a sample may be detected and quantified using Western blot analysis.
- the technique generally comprises separating sample products by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with labeling antibodies that specifically bind to the analyte protein.
- the labeling antibodies specifically bind to analyte on the solid support.
- labeling agents such as antibodies (e.g., labeled sheep anti-mouse antibodies where the antibody to an analyte is a murine antibody) that specifically bind to the labeling antibody.
- the present invention is directed to methods of providing a glucose-responsive insulin-secreting capability to a mammal in need of such capability, such as an insulin-deficient mammal or a diabetic human.
- These methods generally comprise injecting or implanting into such a mammal engineered cells which secrete insulin in response to glucose.
- encapsulation involves the polymerization of one or more matrix precursors in the presence of the cells to be encapsulated.
- the matrix precursors must be non-toxic, biocompatible, and must promote rapid polymerization.
- polymerization for the purposes of this application, is defined to include any reaction by which soluble precursor materials are transformed to a shape-retaining, insoluble form, including chain formation, increase in chain length, and covalent or non-covalent cross-linking.
- Matrix precursors may include alginates, polylysine, chitosan plasma, fibrinogen, casein, fibrin, limulus lysate, milk protein, collagen, agarose, carrageenan, agar, guar gum, gum arabic, pectin, tragacanth gum, xanthan gum, and mixtures thereof.
- a first aqueous solution containing matrix precursors is contacted with a second aqueous solution containing the cells.
- the concentration of matrix precursor for most matrices can be from 1 microgram/ml to 100 mg/ml, preferably from 50 microgram/ml to 20 mg/ml and optimally from 0.1 mg/ml to 6 mg/ml.
- the second solution may contain substances that promote polymerization, or these may be added in a third aqueous solution.
- Substances which promote polymerization may comprise multivalent ions in solution which can form a salt with matrix monomers.
- the optimum ion is a physiologically compatible ion such as calcium in a concentration which provides in the mixture of the first and second solution, a calcium ion concentration which promotes rapid polymerization of the gel polymer precursor.
- the formed matrix particles can have a particle size as low as 0.02 mm in diameter and a size as large as 3 mm. Other ranges may be preferred and optimum for matrices having different matrix strengths, rigidity and different cell attachment characteristics.
- the primary solution can be dripped or blown under air pressure into the secondary solution, the drop size being selected to yield the desired particle size.
- a distribution of particles can be obtained with certain gel precursors by subjecting the secondary mixture to agitation while the primary solution is rapidly poured into the secondary solution.
- the two solutions can be quickly mixed and the mixture placed in a cavity having the general configuration of the desired product.
- Pore diameters range from 5 x 10 3 to 40 microns, preferably from 2 x
- the final porosity (average pore diameter, pore size distribution, average pore length, and total pore numbers) of the matrix particles is dependent upon the initial concentration of the gel polymer in the particle, the degree of gel polymer crosslinking obtained, the degree of spatial contraction occurring during matrix polymerization, and other variables, including processing temperatures.
- the matrix preferably has a porosity which allows only substances of molecular weight less than about 100,000 Daltons to pass transversely there-through while carrying blood along the length of the fiber.
- Substances of molecular weight below this cutoff including substances which stimulate insulin secretion, such as glucose, amino acids, fatty acids, hormones (e.g., thyroxine, growth hormone, glucocorticoids) , and neuronal stimuli diffuse through the hollow fiber wall to the islets. In response to these substances, the islets produce insulin, which diffuses through the matrix into the bloodstream.
- substances which stimulate insulin secretion such as glucose, amino acids, fatty acids, hormones (e.g., thyroxine, growth hormone, glucocorticoids)
- hormones e.g., thyroxine, growth hormone, glucocorticoids
- the matrix can have any desired cell density.
- the cell density can range from 10 3 to 5 x 10 8 cells/ml of matrix.
- the formed matrix and enclosed cells are placed in a nutrient medium for maintaining the viability of the cells.
- a nutrient medium for maintaining the viability of the cells.
- the nutrients required for maintenance and propagation of various cells are generally well established and fully within the knowledge and skill of the person skilled in the art.
- suitable media are described in READINGS IN MAMMALIAN CELL CULTURE, Robert Pollack (editor), Second edition New York: Cold Spring Harbor Laboratory ( 1 981 ), and in other publications. Further processing of the matrix to preserve or to expand or multiply the cells may be desirable, depending upon the cell type and factors involved in the insertion.
- the cells embedded within matrix particles may be implanted in a mammal.
- the matrix is preferably implanted in a portion of the body which is not functionally affected by the implant and which will readily vascularize the implant.
- pancreatic islet cells that are enclosed in a matrix can be implanted in the peritoneum, mesenteric omentum, kidney subcapsular space, splenic pulp, portal vein, and other sites appropriate for vascularization and function.
- the implanted engineered cells of this invention remain encapsulated.
- the fugitive cells may be destroyed by exposure to drugs to which the cells are sensitive.
- the engineered cells of this invention may be manipulated to encode and express, constitutively or inducibly, a drug sensitivity gene such as cytosine deaminase. Any cells that escape their capsule may be exposed to 5-fluorocytosine, which is converted to the toxic metabolite 5 fluorouracil by fugitive cells and their offspring. The fugitive cells then die. Surrounding tissue cells that do not express the cytosine deaminase gene will not be killed.
- unencapsulated glucose-sensitive cells of this invention may be implanted into a host animal. Implantation by this approach may circumvent problems with viability or function, at least for the short term, that may be encountered with the encapsulation strategy. This approach allows for testing of the function of the cells in experimental animals but is not preferred as a strategy for treating human patients.
- G. Gene therapy with engineered cells that express calbindin and secrete insulin in a glucose-sensitive fashion
- insulin-producing cells are taken from a mammal that does not secrete insulin in a glucose-sensitive fashion, optionally immortalized, transduced with a nucleic acid that encodes calbindin and, optionally, with a nucleic acid that encodes a drug sensitivity, and re-introduced into the mammal. This approach minimizes potential problems with immune rejection.
- Pancreatic islet cells suitable for engineering and re-implantation can be derived from pancreatic tissue by numerous published procedures or they can be derived from tissue, organ or cell cultures. Procedures for preparing pancreas tissue for transplant are described by Lafferty ef al. ( 1 984), Transplantation Proceedings 14: 71 4 and by Lacy ef al. ( 1 984), Ann. Rev. Immunol. 2: 1 83. Briefly, pancreatic tissue is infused via the pancreatic duct with a solution of an enzyme such as collagenase which digests connective tissue and disrupts the integrity of the gland. The gland is further dissociated by shaking with marbles until tissue fragments are reduced to a size of less than 500 microns diameter.
- an enzyme such as collagenase which digests connective tissue and disrupts the integrity of the gland.
- This disassociation procedure releases islets from the exocrine tissue that surrounded them. Islets are then separated from non-islet tissue by centrifugation on a discontinuous gradient such as one made of Ficoll TM (Pharmacia Fine Chemicals, Inc.) (27% w/v; 23.5% w/v; and 1 1 % w/v) .
- Ficoll TM Pulcoa Fine Chemicals, Inc.
- Primary isolates (or primary cultures) of islet cells in culture may be directly transduced as described above with vectors that contain a gene for calbindin and, optionally, a drug sensitivity gene, and implanted as described above.
- the cells to be implanted by the methods of the present invention can optionally be immortalized by a variety of techniques known in the art, which include transformation with Epstein-Barr virus or with retroviruses, or the transfection of oncogenes.
- immortalization by Epstein-Barr virus may be employed, as described in U.S. Patent No. 4,464,465, incorporated herein by reference.
- Epstein-Barr virus mutants which lack OriP and OriLyt origins of replication are particularly useful.
- pancreatic islet cells for implantation is fetal pancreatic proislet cells that are transduced with a nucleic acid that encodes a calbindin molecule. These can be derived from the recipient species (con- specific implantation) or they can be derived from a donor species which is different from the recipient species. When implanted into a suitable vascularized site in the body, the cells differentiate to produce islet cells in a vascularized environment, an artificial endocrine pancreas, which responds to serum glucose by producing and secreting insulin.
- Example 1 Production of glucose-sensitive engineered cells
- Rat insulinoma cells (RIN) 1 046-38) were transduced with pREP- calbindin-D 28k and cloned as follows:
- Full length calbindin-D 28k (CaBP 28k ) cDNA (lacking the 5' and 3' untranslated region) is isolated by PCR from cDNA prepared from rat renal distal tubular mRNA.
- the PCR product is purified, digested with Not I and Sfi I and ligated into the eukaryotic expression vector pREP 4 (Invitrogen) downstream of the RSV promoter.
- the plasmids pFOXCATI .SWT, pFOXCAT2.RIP1 -85 and pFOXCAT2.RIP1 -85.FF5 were kindly provided by Dr.
- the CaBP probe for Northern analysis was a 1 7 bp fragment obtained by EcoR I digestion of the Bluescript plasmid.
- the insulin probe was a 0.68 kb mouse insulin insert present in the Pstl-EcoRI site of pGEM.
- the 2.1 kb chicken ⁇ -actin probe was a Hind III digest of pBR322 plasmid and the cDNA for 1 8S rRNA came from Ramareddy Guntaka (University of Missouri at Columbia) .
- Pancreatic islet cells are readily infectible by adenoviruses (Efrat et al. , P. N.A.S. 92:6947, 1 995).
- an adenoviral vector is used to introduce calbindin into islet cells.
- a replication incompetent adenovirus vector expressing calbindin is constructed as follows.
- a calbindin-encoding nucleic acid is cloned into a conventional plasmid such as bluescript, pgem, pUC or pBR322 as a carrier, together with (from 5' to 3):
- Cytomegalovirus immediate early enhancer nt 1 88-843 of M64943 as found in GenBank: GGCACATGGCCAATGCATATCGATCTATACATTGAATCAATATTGGCCATTAG
- Sequences I-V are operably linked in the prescribed order by suitable restriction and ligation reactions. Together, these comprise a calbindin-adenovirus 5 shuttle vector.
- HEK 293 cells are co-transfected into HEK 293 cells using calcium phosphate transfection.
- the cells are overlaid with soft agarose and after 5-8 days, individual lytic plaques identified by inspection after neutral red staining.
- the agarose plugs containing viruses are picked, expanded and virus is prepared by infecting HEK 293 cells and purifying virus by CsCl banding.
- pREP4 Invitrogen
- D28K calbindin
- Herpes Simplex Virus thymidine kinase Three nucleic acid fragments are cloned into the pREP4 (Invitrogen) expression vector to yield a bicistronic expression cassette expressing both calbindin (D28K) and the Herpes Simplex Virus thymidine kinase.
- the first fragment is the coding region of the Herpes Simplex Virus thymidine kinase gene (GenBank accession number: V00467; M. J. Wagner, J .
- PCR primer is 5'gatcaCCatggcttcgtacccctgcc
- BamHl site on the 3' end
- Fragment 2 is the IRES fragment from the encephalomyocarditis virus which is obtained from the commercially available plasmid pCITE-1 (Novagen) by PCR.
- Fragment 3 is the calbindin cDNA which is modified by PCR to have a Hindlll site on its 5' end along with a KOZAK consensus sequence, and a ⁇ /ofl site on its 3' end.
- the 5' PCR primer used is agctaagcttCCACCatggcagaatcccacctg
- the 3' PCR primer used is ACTCGCGGCCGCctagttgtccccagcagag.
- the vector pREP4 is digested with Xhol and BamHl.
- the HSV-Tk gene (fragment 1 ) is digested with BamHl and Ncol.
- the IRES fragment (fragment 2) is digested with Xhol and Ncol. These fragments are combined in a standard trimolecular ligation to yield an intermediate plasmid designated pREP4-IRES-tk.
- the intermediate plasmid pREP4-IRES-tk is digested with Hindlll and NotI.
- Fragment 3 containing the calbindin gene is digested with Hindlll and NotI. These are ligated together to yield the plasmid pREP4-CB-IRES-tk.
- RIN 1 046-38 cells grown in RPMI 1 640
- T-75 flasks are used to carry out stable transduction by lipofection.
- 1 0 ⁇ g each of pREP 4 (vector alone) and pREP-CaBP 28k (calbindin over-expressing) or pREP4-CB-IRES-tk are mixed well in separate tubes with 50 to 75 ⁇ l of lipofectin and this mixture is added to the cells (in 5 ml of serum-free media) and incubated for 1 8 h at 37 °C. Media with serum is added to the cells the next day.
- the proteins are then transferred onto an Immobilon-P membrane, the membrane was blocked using 2% BSA in 1 X PBS and a 1 :4 dilution of normal goat antisera in 2% BSA/PBS. After washing the membrane with PBS, the membrane is incubated with rabbit anti-rat calbindin antisera overnight at 4°C with 125 l-labeled protein A/2% BSA/PRS/0.05% Tween-20, then washed thoroughly with PBS and air dried before autoradiographic exposure.
- Calbindin levels are determined by radioimmunoassay (RIA) using antiserum against rat renal calbindin and using rat renal CaBP as standard
- RNA was isolated from various clones by the guanidinium isothiocyanate/phenol/chloroform method (Chomzynski and Sacchi ( 1 987), Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1 62: 1 5-1 59) and poly(A) + RNA was selected via oligo(dT) cellulose affinity chromatography. Equivalent amounts poly(A) + RNA were separated under denaturing conditions on a 1 .2% formaldehyde agarose gel and transferred to a nylon filter in 20% SSC.
- the filters were baked at 80°C for 2-3 h, prehybridized, hybridized and washed using 1 -3 x 10 6 cpm/ml 32 P labeled cDNA/filter.
- cDNA was labeled to a specific activity of 10 8 -1 0 9 cpm/ ⁇ g DNA by the oligonucleotide labeling procedure using random primed DNA labeling kit (Boehringer-Mannheim).
- the filters were probed with insulin, ⁇ -actin and 1 8S cDNA after stripping using a solution that contained 50% formamide, 6 x SSC at 65 ° C/30 min. between each probing.
- Fig. 1 Five positive clones were found to overexpress calbindin as determined by Western blot analysis (Fig. 1 ).
- the positive clones were the ones numbered C2, C1 0, C 1 1 , C1 3 and C5.
- the clones were plated in 1 2-well plates with 2-3 x 1 0 5 cells/well and after 2-4 days of culture, the medium was removed. Fresh media was added to the culture and incubated for 1 h before collecting samples for insulin assay. Insulin was determined by RIA with a dextran-charcoal method using a rat insulin standard (Novo Laboratories, Danbury, Connecticut) and guinea pig anti-insulin serum (Clark, 1 990) .
- 125 l labeled insulin was obtained from DuPont-New England Nuclear. To determine insulin content of clones, cells were plated as for secretion studies and after two days cells were rjnsed with PBS, acid-ethanol with 50 mM benzamidine was added to the wells (0.5 ml) and the wells were sealed with pressure sensitive film. The extract was collected after 48 h at 4°C diluted > 10 times with assay buffer, then frozen at -20°C for subsequent insulin assays.
- the average concentration of calbindin in cells transduced with vector alone was 0.4 ⁇ 0.1 ⁇ g/mg protein.
- Clone C2 expressed the highest level of calbindin ( 14.2 ⁇ 1 .7 ⁇ g/mg protein) which represented a 35 fold induction in calbindin.
- Clone C1 0 expressed the next highest level of calbindin (3.7 ⁇ 0.4 ⁇ g/mg protein) followed by C 1 3, C5 and C1 1 .
- Table I positive clones were found to overexpress calbindin from 6.5 fold (for C 1 1 ) to a higher level of overexpression of 35 fold for C2.
- Example 3 Construction and use of artificial tissue comprising engineered calbindin-expressing glucose-sensitive cells
- the engineered cells of the present invention are implanted, for example, using the alginate-polylysine encapsulation technique of O'Shea and Sun ( 1 986), Diabetes 35: 943-946, as modified by Fritschy et al. ( 1 991 ), Diabetes 40: 37.
- Monolayers of cultured engineered calbindin-expressing insulinoma cells are harvested by trypsinization and washed twice in isotonic, neutral pH buffer. The number of cells and the percent viability are determined by trypan blue exclusion with a hemocytometer. Viable cells are maintained on ice and centrifuged ( 1 500 x g) for 5 min before use, and the supernatant is discarded.
- the collected engineered cells are suspended in 0.1 - 1 .3% sodium alginate and encapsulated by extrusion of drops of the cell/alginate suspension through a syringe into CaCI 2 . After several washing steps, the droplets are suspended in 0.1 -3% polylysine or chitosan and rewashed. The alginate within the capsules is then reliquified by suspension in 1 mM EGTA and then rewashed with Krebs balanced salt buffer. Each capsule contains several hundred cells and has a diameter of approximately 1 mm.
- Kelco Corp. is wrapped in aluminum foil and autoclaved for 1 5 minutes.
- a 1 % solution of calcium sodium alginate in cell culture medium is prepared at 35-40° C.
- about 10 8 engineered cells are pelleted by gentle centrifugation.
- the culture medium is decanted from the pelleted islets and approximately 1 4 ml of the alginate/medium solution is added.
- the cell mixture is centrifuged at about 5000 x g for five minutes to form two layers.
- the bottom layer contains a viscous alginate and cell suspension and the top layer consists of the culture medium.
- the culture medium is removed by pipetting, and the remaining encapsulated cell suspension is transferred to a syringe.
- the encapsulated cells are injected intraperitoneally into a suitable recipient that has an impaired ability to secrete insulin in a glucose-sensitive fashion.
- the blood glucose levels of the recipient are monitored by methods known in the art to ensure that glucose homeostasis is maintained.
- the total number of cells to be implanted in a given recipient vary as a function of the recipient's body mass, metabolic needs, innate ability to secrete insulin and innate ability to control glucose. In general, the number of implanted cells should be sufficient to maintain glucose homeostasis.
- the accumulated data from human and animal experiments indicates that the number of islets generally required to compensate for impaired insulin secretion is about 5,000 islets/kg body weight. Thus, a 70 kg patient would need approximately 350,000 islets to maintain suitable blood glucose levels, or between about 10 5 and about 1 0 10 encapsulated cells.
- ORGANISM Rattus norvegicus
- F TISSUE TYPE
- CTGCAGTCAT CTCTGATCAC AGCCTCACAG TTTTTTGAGA TCTGGCTTCA TTTCGACGCT 360 GATGGAAGTG GTTACCTGGA AGGAAAGGAG CTGCAGAACT TGATCCAGGA GCTTCTGCAG 420
- TGTGGCACAT TCTTTTCTGC TTGTTTCTAT ACTGTTTGTA ATGTACAGTT TTTGTAAGCA 1260 TATAATTGAA AAGAAGAAAG TCTATGCTTA GGCCAGTCAG TATAATCCAT TTTCAAAGAT 1320
- ORGANISM Homo sapiens
- F TISSUE TYPE
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AU74557/96A AU7455796A (en) | 1995-10-18 | 1996-10-17 | Method for restoring glucose responsiveness to insulin secretion |
US09/051,696 US6319495B1 (en) | 1995-10-18 | 1996-10-17 | Method for restoring glucose responsiveness to insulin secretion |
EP96936701A EP0859640A4 (en) | 1995-10-18 | 1996-10-17 | Method for restoring glucose responsiveness to insulin secretion |
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US538695P | 1995-10-18 | 1995-10-18 | |
US60/005,386 | 1995-10-18 |
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EP0707646A1 (en) * | 1993-06-28 | 1996-04-24 | The Board Of Regents, The University Of Texas System | Vectors for genetically engineered cells that produce insulin in response to glucose |
-
1996
- 1996-10-17 CA CA002233549A patent/CA2233549A1/en not_active Abandoned
- 1996-10-17 US US09/051,696 patent/US6319495B1/en not_active Expired - Fee Related
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- 1996-10-17 EP EP96936701A patent/EP0859640A4/en not_active Withdrawn
Non-Patent Citations (6)
Title |
---|
HUSAIN S M, ET AL.: "EFFECT OF DIABETES MELLITUS ON MATERNOFETAL FLUX OF CALCIUM AND MAGNESIUM AND CALBINDIN9K MRNA EXPRESSION IN RAT PLACENTA", PEDIATRIC RESEARCH, LIPPINCOTT WILLIAMS & WILKINS, NEW YORK, US, vol. 35, no. 03, 1 March 1994 (1994-03-01), US, pages 376 - 381, XP001052769, ISSN: 0031-3998 * |
LEE SOOJA ET AL: "1,25-Dihydroxyvitamin D-3 and pancreatic beta-cell function: Vitamin D receptors, gene expression, and insulin secretion.", ENDOCRINOLOGY, THE ENDOCRINE SOCIETY, US, vol. 134, no. 4, 1 January 1994 (1994-01-01), US, pages 1602 - 1610, XP002179618, ISSN: 0013-7227, DOI: 10.1210/en.134.4.1602 * |
NIETO-BONA M P, BUSIGUINA S, TORRES-ALEMAN I: "INSULIN-LIKE GROWTH FACTOR I IS AN AFFERENT TROPHIC SIGNAL THAT MODULATES CALBINDIN-28KD IN ADULT PURKINJE CELLS", JOURNAL OF NEUROSCIENCE RESEARCH., WILEY-LISS., US, vol. 42, no. 03, 15 October 1995 (1995-10-15), US, pages 371 - 376, XP001052770, ISSN: 0360-4012, DOI: 10.1002/jnr.490420311 * |
RHOTEN W B, SERGEEV I N: "CALBINDIN-D28K APPEARS TO BUFFER INTRACELLULAR CA2+ IN BUTYRATE-TREATED RAT INSULINOMA CELL LINE", ENDOCRINE, HUMANA PRESS, INC., US, vol. 02, no. 11, 1 November 1994 (1994-11-01), US, pages 989 - 995, XP001052768, ISSN: 1355-008X * |
See also references of EP0859640A4 * |
YAMAGUCHI T, KEINO K, FUKUDA J: "THE EFFECT OF INSULIN AND INSULIN-LIKE GROWTH FACTOR-I ON THE EXPRESSION OF CALRETININ AND CALBINDIN D-28K IN RAT EMBRYONIC NEURONS IN CULTURE", NEUROCHEMISTRY INTERNATIONAL., PERGAMON PRESS, OXFORD., GB, vol. 26, no. 03, 1 March 1995 (1995-03-01), GB, pages 255 - 262, XP001052771, ISSN: 0197-0186, DOI: 10.1016/0197-0186(94)00127-G * |
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US6319495B1 (en) | 2001-11-20 |
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