Title:  Encapsulated cells containing an amplified expression vector as a drug delivery device

United States Patent:  6,713,293

Issued:  March 30, 2004

Inventors:  Grummt; Friedrich (Walther-von-der-Vogelweide Strasse 55, Wurzburg, DE 97074); Sauer; Birgitta (Meichelbeek Strasse 2, Penzberg, DE 82377); Muller; Martha (Ursulinengasse 15, Wurzburg, DE 97070)

Appl. No.:  246309

Filed:  February 8, 1999

Abstract

The present invention provides a drug delivery device comprising encapsulated cells that contain multiple copies of an expression cassette. The drug delivery device of the present invention is useful to supply an animal, including a human, with a therapeutically desirable molecule, including physiological activities lacking in disease conditions or antagonist against conditions in an animal, especially a human.

SUMMARY OF THE INVENTION

The present invention provides a drug delivery device ("device") and methods of making and using such a device. The device of the present invention is useful for many disease control and therapeutic strategies by taking advantage of the ability of a cell to provide pharmaceutically useful functions, for example the metabolism of a drug precursor. Thus, the device of the invention can, once transferred into a diseased animal, provide long term disease control.

In one embodiment, the device of the present invention comprises a eukaryotic cell that contains an exogenous polynucleotide ("exogenous polynucleotide") sequence, preferably in multiple copies, and that is located in a capsule. In one aspect, the exogenous polynucleotide comprises an amplification-promoting sequence and an expression cassette. In another aspect, the exogenous polynucleotide also comprises a selectable marker.

In one embodiment, an amplification-promoting sequence ("APS") useful for the exogenous polynucleotide in a device of the present invention, is capable of amplifying the number of copies of the exogenous polynucleotide in a eukaryotic cell. In a preferred aspect, an APS presented in SEQ ID NO:1 or SEQ ID NO:2 is used. In a further aspect, any polynucleotide that is substantially homologous to SEQ ID NOS:1 or 2 is also a useful APS in the exogenous polynucleotide. In another aspect, more than one APS is included in the exogenous polynucleotide, for example SEQ ID NOS:1 and 2. In a further aspect, one or more APSs are included in the exogenous polynucleotides, of which one or more is substantially homologous to either SEQ ID NOS:1 or 2 and of which one or more is identical to SEQ ID NOS:1 or 2.

An expression cassette useful in the exogenous polypeptide, in one embodiment, comprises a polynucleotide sequence that encodes a function of interest ("coding sequence") and a regulatory sequence. The coding sequence in the expression cassette, in one aspect, may encode a peptide, a polypeptide, a protein, a polynucleotide. In another aspect, the expression cassette may be specific for a polynucleotide or an oligonucleotide with a catalytic or an inhibitory activity. Preferably, the coding sequence is specific for a molecule that has a pharmacologically desirable activity or property. In another aspect, the coding sequence encodes a protein or a polypeptide with an enzyme activity that is lacking in a disease condition, for example an enzyme activity involved in the synthesis of a signal transduction molecule (e.g., a second messenger, serotonin, dopamin). In another aspect, the coding sequence is specific for a peptide, a polypeptide or a protein capable of regulating cell proliferation, cell differentiation, programmed cell death, signal transduction, gene expression, gene transcription, translation, etc. In another aspect the coding sequence is specific for aft antibody, an antibody fragment, an agonist of a naturally occurring molecule, an antagonist of a naturally occurring molecule, a neurotrophic factor, a neurotropic factor, a peptide hormone (e.g., a neurohormone), etc.

In another preferred embodiment, the coding sequence is specific for tyrosine hydroxylase, tryptophan hydroxylase, melanoma inhibitory activity, an insulin, an insulin precursor polypeptide, an enkephalin, an enkephalin precursor polypeptide, parathyroid hormone, a parathyroid hormone precursor polypeptide, enkephalinase, a neurotrophic factor, a neurotropic factor, or a homolog, orthologue, paralogue, analogue, or derivative of any of these, or a fusion polypeptide or protein that contains any of the above and a heterologous protein, polypeptide or peptide.

Preferably, the expression cassette contains a regulatory sequence. In one aspect, the expression cassette contains a promoter sequence and a polyadenylation sequence ("polyA sequence"). Promoter sequences useful for the expression cassette, in a preferred aspect, are active, i.e., capable of initiating gene transcription, in the cell type used in the device of the present invention. Useful promoters are active at least at a moderate level (i.e., a moderately strong promoter), preferably they are active at a high level (i.e., strong promoter). A constitutive and an inducible promoter may be used in the expression cassette, provided the promoter is sufficiently active. In another aspect, any polyA sequence that is sufficiently strong to induce polyadenylation of the transcripts of the expression cassette can be used.

In another aspect, the expression cassette may include an enhancer, an intron, a 5' and/or 3' untranslated region, an RNA stability regulating sequence or a combination of more than one of these elements.

In one embodiment, a eukaryotic cell is used in the device of the present invention. The cell preferably is capable of dividing multiple times, for example about 10 to about 300 times, or more than 300 times. In one aspect, the cell is transformed. In another aspect, the cell is derived from the same species as the species in which the device of the present invention is used.

In one embodiment, the cell containing the exogenous polynucleotide is encapsulated. In a preferred aspect, the capsule that contains the cell is double layered. In a preferred aspect, the cells of the device of the present invention are located in the inner layer. In another aspect, the capsule has more than two layers.

DETAILED DESCRIPTION OF THE INVENTION

5.1. Overview

The present invention relates to drug delivery devices that take advantage of the ability of most eukaryotic cells to synthesize or aid in the synthesis of molecules. The lack of one or more of these molecules is often responsible for a disease condition in an animal, for example a human. Thus, when properly supplied as drugs to a diseased animal, these molecules are useful in controlling and/or treating many disease conditions.

In one embodiment, the present invention provides drug delivery devices that are capable of synthesizing a molecule, for example a molecule having a pharmacological activity. When the device is transferred into an animal, the molecule is provided to that animal by the device of the present invention, i.e., the device acts as a drug pump which produces or aids in producing a drug.

The device of the present invention comprises encapsulated eukaryotic cells which contain an exogenous polynucleotide (i.e., exogenous to the cells). The exogenous polynucleotide contains an expression cassette that includes a coding sequence specific for a metabolic activity, for example an enzyme activity that is lacking in a disease condition. See, e.g., U.S. Pat. No. 5,082,670, which provides examples of such metabolic activities. The expression cassette further contains a promoter and a polyA sequence to regulate the proper transcription of the coding sequence. The exogenous polynucleotide can be constructed with any technique known in the art of molecular biology. For general background on molecular biology techniques, see, e.g., Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York; Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, New York; and U.S. Pat. No. 5,650,148.

The exogenous polynucleotide also contains an APS capable of amplifying the copy number of the exogenous polynucleotide in the genome of eukaryotic cells. Thus, the cells of the device of the present invention contain multiple copies of an expression cassette specific for a metabolic activity. As demonstrated in the Examples below, the expression of the coding sequence of the exogenous polynucleotide increases with an increase in the number of copies of the exogenous polynucleotide in the cells, i.e., gene dosage effect. This increased expression provides the metabolic activity supplied in the device of the present invention at a higher level and therefore makes the device a more potent drug pump than without amplification of the exogenous polynucleotide.

The exogenous polynucleotide used in the device of the present invention is up to about 50 kilobases ("kb") in size, preferably up to about 40 kb, more preferably up to about 30 kb and most preferably up to about 20 kb.

5.2. Amplification-Promoting Sequences

APSs useful for the device of the present invention are capable of amplifying (i.e., increasing) the number of copies of a polynucleotide sequence in a cell. For example, when an APS is integrated into an exogenous polynucleotide that is transfected into a proliferating cell, the number of copies of the exogenous polynucleotide will increase with increasing numbers of divisions of that cell. The exogenous polynucleotide will typically integrate into the genome of the cell following transfection. See, e.g., Hemann et al., 1994, DNA and Cell Biology 13:437-445; Meyer et al., 1993, Gene 129:263-268; Wegner et al., 1989, Nucleic Acids Research 17:9909-9932, which discuss APSs and their use.

The rate of amplification of the exogenous polynucleotide containing an APS useful for the device of the present invention can be expressed through the amplification factor, i.e., the number of copies of the exogenous polynucleotide found in a single host cell after 15 to 25 divisions of the host cell following transfection. An APS useful for the device of the present invention has an amplification factor of at least about 5, more preferably at least about 10, more preferably at least about 20, more preferably at least about 40, more preferably at least about 70, more preferably at least about 100 and most preferably at least about 150. See, e.g., U.S. Pat. Nos. 5,756,455 and 4,442,203, which discuss gene amplification and methods for its detection.

In one embodiment, one APS is included in the exogenous polynucleotide used in the device of the present invention. In another embodiment, more than one APS, for example two, are included in the exogenous polynucleotide.

In a preferred embodiment, an APS used in the device of the present invention has a sequence as presented in SEQ ID NO:1 or SEQ ID NO:2 (See, Wegner et al., 1989, Nucleic Acids Research 17:9909-9932) (Genbank accession numbers X52413 and X52412, respectively). In another preferred embodiment, an APS used in the device of the present invention has a sequence that is at least about 85% identical to a polynucleotide sequence as presented in SEQ ID NO:1 or SEQ ID NO:2, more preferably at least about 90%, more preferably at least about 95% and more preferably at least about 98%.

In another preferred embodiment, an APS useful for the device of the present invention is a polynucleotide that hybridizes under moderately stringent conditions to a polynucleotide that is complementary to SEQ ID NO:1 or SEQ ID NO:2. Moderately stringent hybridization conditions are well known in the art. For example, filters containing DNA are pretreated for 6 h at 55^oC. in a solution containing 6xSSC, 5xDenhart's solution, 0.5% SDS and 100 .mu.g/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution and 5-20x10⁶ cpm ³² P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 55^oC. The filters are then washed in approximately 1x wash mix (10x wash mix contains 3M NaCl, 0.6M Tris base, and 0.02M EDTA) twice for 5 minutes each at room temperature, then in 1x wash mix containing 1%SDS at 60^oC. for about 30 min, and finally in 0.3x wash mix containing 0.1% SDS at 60^oC. for about 30 min. The filters are then air dried and exposed to x-ray film for autoradiography.

In an alternative protocol, washing of filters is done twice for 30 minutes at 60^oC. in a solution containing 1xSSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography.

Other conditions of moderate stringency which may be used are also well-known in the art, for example the washing of filters can be done at 37^oC. for 1 h in a solution containing 2xSSC, 0.1% SDS. Another example of hybridization under moderately stringent conditions is washing in 0.2xSSC/0.1% SDS at 42^oC. (Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York).

In a more preferred embodiment, an APS useful for the device of the present invention is a polynucleotide that hybridizes under highly stringent conditions to a polynucleotide that is complementary to SEQ ID NO: 1 or SEQ ID NO:2. Hybridization under highly stringent conditions is well known in the art. For example, prehybridization of filters containing DNA to be screened is carried out for 8 h to overnight at 65^oC. in a buffer containing 6xSSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 .mu.g/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65^oC. in prehybridization mixture containing 100 .mu.g/ml denatured salmon sperm DNA and 5-20x10⁶ cpm of ³² P-labeled probe. The filters are then washed in approximately 1x wash mix (10x wash mix contains 3M NaCl, 0.6M Tris base, and 0.02M EDTA) twice for 5 minutes each at room temperature, then in 1x wash mix containing 1% SDS at 60^oC. for about 30 min, and finally in 0.3x wash mix containing 0.1% SDS at 60^oC. for about 30 min. The filters are then air dried and exposed to x-ray film for autoradiography. In an alternative protocol, washing of filters is done for 37^oC. for 1 h in a solution containing 2xSSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1xSSC at 50^oC. for 45 min before autoradiography. Another example of hybridization under highly stringent conditions is hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65^oC., and washing in 0.1xSSC/0.1% SDS at 68^oC. (Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3).

5.3. Expression Cassettes

The exogenous polynucleotide used in the device of the present invention contains an expression cassette. The expression cassette contains a polynucleotide sequence specific for a metabolic activity, ie., coding sequence. The expression cassette further contains regulatory elements for the expression of the coding sequence, i.e., a promoter and a polyA sequence. Other regulatory elements that may be used in the expression cassette include an enhancer, an intron, 5' and/or 3' untranslated regions, RNA stability regulating sequences, etc.

5.3.1. Coding Sequences

The coding sequence is a polynucleotide sequence found in the exogenous polynucleotide of the present invention specific for a metabolic activity, i.e., any activity relevant to the biochemistry, physiology or pharmacology of the recipient animal. The coding sequence may encode a protein or a nucleotide factor.

The coding sequence in the expression cassette, in one aspect, encodes a peptide, a polypeptide, a protein, a polynucleotide or an oligonucleotide. Preferably, the coding sequence is specific for a molecule that has a pharmacologically desirable activity or property. In another aspect, the coding sequence encodes a protein or a polypeptide with an enzyme activity that is lacking in a disease condition, for example an enzyme activity involved in the synthesis of a signal transduction molecule (e.g., a second messenger, serotonin, dopamin). In another aspect, the coding sequence is specific for a peptide, a polypeptide or a protein capable of regulating cell proliferation, cell differentiation, programmed cell death, signal transduction, gene expression, gene transcription, translation, etc. In another aspect the coding sequence is specific for an antibody, an antibody fragment, an agonist of a naturally occurring molecule (e.g., an agonist of a receptor molecule in the recipient animal), an antagonist of a naturally occurring molecule (e.g., an antagonist of a receptor molecule in the recipient animal), a neurotrophic factor, a neurotropic factor, a peptide hormone (e.g., a neurohormone), etc.

In another aspect, the coding sequence may be specific for a non-naturally occurring molecule, e.g., protein, polypeptide, peptide, polynucleotide, oligonucleotide, etc. The device of the present invention is useful for the delivery of any molecule that can be provided through expression of a polynucleotide sequence in the expression cassette of the device and the molecule may be of any origin, whether natural or nonnatural. The purpose for expressing a particular polynucleotide sequence in the expression cassette of the device is of concern to the overall strategy pursued with the use of the device. However, the device itself is not limited in any way to particular classes of molecules that may be provided to the recipient animal through the device.

Proteins that may be encoded by the coding sequence include, but are not limited to, enzymes, precursor proteins and polypeptides of signal transduction peptides and polypeptides, growth factors, neurohormones, toxins, neurite promoting molecules, neurotropic factors and neurotrophic factors (e.g., brain derived neurotrophic factor, ciliary neurotrophic factor, nerve growth factors, etc.); enzymes involved in the synthesis of gangliosides, antibiotics, idenosin phosphates, cyclic adenosin monophosphate, cyclic guanosin monophosphate, nucleoside mono-, di- or triphosphates, steriods, glucosides, glycerides, catecholamines, endorphins, peptides, etc. See, e.g., U.S. Pat. Nos. 5,800,828; 5,798,113; 5,762,926; 5,656,481; 5,653,975; 5,650,148; 5,082,670, which discuss coding sequences and metabolic activities that can be expressed using the device of the present invention.

In another preferred embodiment, the coding sequence is specific for tyrosine hydroxylase, tryptophan hydroxylase, melanoma inhibitory activity, an insulin, an insulin precursor polypeptide, an enkephalin, an enkephalin precursor polypeptide, parathyroid hormone, a parathyroid hormone precursor polypeptide, enkephalinase, a neurotrophic factor, a neurotropic factor, or a homolog, orthologue, paralogue, analogue, or derivative of any of these, or a fusion polypeptide or protein that contains any of the above and a heterologous protein, polypeptide or peptide.

When choosing a coding sequence for the device of the present invention, it should be considered that the molecule of interest that is generated through expression of the coding sequence must be capable of leaving, e.g., diffusing out of, the capsule used in the device. For example, when the coding sequence is specific for a precursor to a peptide (e.g., a neurotransmitter, a peptide hormone, etc.), the permeability barrier of the capsule should be such that the peptide can leave the capsule, or such that the precursor can leave the capsule in case the precursor is not processed in the cells of the device of the present invention.

When the coding sequence is specific for an enzyme, the substrate of the enzyme should be available to that enzyme, for example through metabolic processing of precursors to the substrate molecule by other enzymes in the cell or through entry of the substrate into the cell. If an enzyme has more than one substrate, that substrate which yields the product of interest through catalysis of the enzyme should be available to the enzyme.

In case the coding sequence is specific for an enzyme that requires a co-factor, the co-factor should be available to the enzyme in the cells of the device of the present invention.

The coding sequence may be specific for a molecule designed to control, prevent or treat a disease, for example metabolic disorders, cancer, diabetes, neurodegenerative diseases, Alzheimer's, neuroregenerative diseases, heart disease, liver diseases, osteoporosis, obesity, arteriosclerosis, etc.

An example of peptides that can be produced using the device of the present invention are enkephalins. Enkephalins are neurohormone peptides that are synthesized in vivo as precursor polypeptides called preproenkephalin and proenkephalin which are processed to yield the enkephalin peptides (see, e.g., Kang et al., 1998, Brain Research 792:133-135; Liu et al., 1996, Journal of Neurochemistry 67:1457-1462). The processing of the preproenkephalin and proenkephalin polypeptide is carried out by peptidases, for example enkephalinase. See, e.g., U.S. Pat. Nos. 5,780,025 and 5,403,585, which describe enkephalinase and methods for its use.

The device of the present invention can be used, for example, to generate enkephalins. For example, one can use a cDNA specific for one or more of the enkephalin precursor peptides and polypeptides in the expression cassette. The enkephalin precursor is preferably expressed in a cell type that is capable of processing the precursor so that one or more mature enkephalin peptides are generated. Another way to generate enkephalins is to express a precursor polypeptide of enkephalins and processing enzymes required to make the individual enkephalin peptides. The particular enzymes needed to process the enkephalin precursors will depend on the cell type used in the device of the invention. Thus, the expression cassette, in this example, would contain more than one coding sequence, i.e., it would contain one coding sequence for the enkephalin precursor and at least one coding sequence for a processing enzyme. Typically, when an expression cassette contains more than one coding sequence, each coding sequence is under the control of its own regulatory elements to direct expression of each coding sequence. When used to express an enkephalin in the recipient animal, especially a human, the device of the invention is preferably located in the lumbar vertebrae.

As another example, the device of the present invention is useful for the generation of serotonin, i.e., 5-hydroxytryptophan. The rate limiting step in the synthesis of serotonin is the hydroxylation of tryptophan by the enzyme tryptophan hydroxylase (see, e.g., Wang et al., 1998, Journal of Neurochemistry 71:1769-1772; Tipper et al., 1994, Arch. Biochem. Biophys. 315:445-453; Kim et al., 1991, Brain Res. Mol. Brain Res. 9:277-283; Hanon et al., 1981, Journal of Physiology 77:269-279). Serotonin can be generated using the device of the present invention by incorporating a polynucleotide sequence specific for tryptophan hydroxylase into the expression cassette of the exogenous polynucleotide.

In another example, the device of the present invention is used to generate insulin. For example, a polynucleotide sequence specific for insulin or an insulin precursor can be used in the expression cassette of the device of the invention. See, e.g., U.S. Pat. Nos. 5,792,656 and 4,914,026, which discuss polynucleotides specific for insulin. The insulin specific polynucleotide may be operatively linked to a promoter that is responsive to induction by elevated concentrations of glucose. See, e.g., Marie et al., 1993, J. Biol. Chem. 268:23881-23890, which describes a glucose inducible promoter. The device of the invention, if designed with a glucose responsive promoter element and an insulin (or insulin precursor) specific polynucleotide, would be capable of generating insulin in the recipient animal when the glucose levels in that animal are elevated. Thus, the device would generate insulin in a glucose dependent manner, as is observed in the islets of Langerhans in the pancreas.

In another aspect, cells capable of increasing insulin production in response to elevated glucose concentration may be provided for the device of the present invention by using a coding sequence specific for a molecule that is part of the glucose sensing mechanism in cells. For example, one may use cells that are capable of expressing and processing insulin and introduce an exogenous polynucleotide into these cells that provides a glucose sensing activity, for example a glucose kinase gene or a glucose transporter gene. See, e.g., U.S. Pat. Nos. 5,792,656 and 5,747,325, which describe cells and polynucleotides useful for this aspect of the present invention.

A further example of using the device of the present invention is the expression of parathyroid hormone ("PTH"). PTH is a peptide hormone involved in the regulation of calcium metabolism. PTH is useful in controlling disease conditions, for example osteoporosis. PTH can further be used to regulate calcium homeostasis. Thus, by using a coding sequence specific for PTH or a PTH derivative, the device of the present invention is useful in controlling pathological and physiological conditions that are amenable to regulation by PTH, for example osteoporosis and calcium homeostasis. See, e.g., Lanske and Kronenberg, 1998, Crit. Rev. Eukaryot. Gene Expr. 8:297-320; Curtis et al., 1997, J. Endocrinol. 154:103-112; Jobert et al., 1996, Mol. Endocrinol. 10:1066-1076; U.S. Pat. Nos. 5,840,837; 5,814,607; 5,783,558; 5,420,242, which provide general background on PTH and which provide polynucleotide sequences for PTH and PTH derivatives useful for the device of the present invention.

5.3.1.1. Expression of Tyrosine Hydroxylase

Tyrosine hydroxylase ("TH") is an enzyme that catalyzes the conversion of tyrosine to dopa through a hydroxylation reaction. Dopa is a precursor of catecholamines which are neurotransmitters and the hydroxylation of tyrosine which results in dopa is the rate limiting step in the synthesis of catecholamins.

A lack of TH activity is observed in Parkinson's disease ("PD"), which is also characterized by the progressive loss of dopaminergic cells (see, e.g., Goetz et al., 1989, Patient Care 23:124-162) Transplantation of dopaminergic or genetically engineered cells expressing efficiently and permanently a functionally TH gene into the brain may provide a means of compensating for the loss of striatal dopamine in PD patients. Using this approach, several investigators have demonstrated partial restoration of abnormalities in rat models (see, e.g., Freed et al., 1992, New England Journal of Medicine 327:1549-1555; Lindvall et al., 1990, Science 247:574-577; Spencer et al., 1992, New England Journal of Medicine 327:1541-1548; Goetz et al., 1989, N. Engl. J. Med. 320:337-341).

Transplantation of cells to treat human diseases such as PD is limited because cells are quickly destroyed by the recipient's immune system. To overcome this limitation, attempts have been made to enclose hormone-secreting cells in a semipermeable membrane that would protect cells from immune attack, yet allow efflux of secretion products (see, e.g., Aebischer et al., 1991, Experimental Neurology 111:269-275; Winn et al., 1991, Experimental Neurology 113:322-329; Tresco et al., 1992, Cell Transplantation 1:225-264; Tseng et al., 1997, Journal of Neuroscience 17:325-333).

A polynucleotide specific for TH was used in the expression cassette of the device of the present invention, see Examples below. See, e.g., U.S. Pat. Nos. 5,300,436 and 5,212,082, which describe polynucleotides encoding various forms of TH and methods of processing and using those polynucleotides, all of which can be used in the device of the present invention.

As demonstrated in the Examples below, an exogenous polynucleotide that contains a TH specific cDNA sequence was amplified in mouse fibroblasts. It was further demonstrated that the exogenous polynucleotide was stably integrated and expressed in the cells when the cells were grown in the absence of selection. It was also shown that TH messenger RNA ("mRNA") was expressed at levels that correlated with the number of copies of the TH cDNA. Thus, the gene-amplification based expression system described herein is useful in providing high levels of TH activity as is desirable for the device of the present invention.

5.3.1.2. Melanoma Inhibitory Activity

Melanoma inhibitory activity ("MIA") is a protein capable of inhibiting the growth of melanoma cells. MIA is a protein of about 131 amino acids in its unprocessed form and about 107 amino acids after removal of a signal sequence. The mature MIA protein is secreted by cells. See, e.g., Bosserhoff et al., 1997, Development Dynamic 208:516-525; van Groningen et al., 1995, Cancer Research 55:6237-6243; Bogdahn et al., 1989, Cancer Research 49:5358-5363 and U.S. Pat. No. 5,770,366 for general background on MIA.

A MIA specific cDNA was incorporated in an exogenous polynucleotide useful for the device of the present invention and the resulting construct was introduced into mouse fibroblasts, as discussed below. As discussed below in the Examples and as shown in FIG. 4, the MIA cDNA was expressed at a high level when using the gene-amplification based expression system described herein.

5.3.2. Regulatory Sequences

The expression cassette used in the device of the present invention contains regulatory sequences designed to express the coding sequence or coding sequences, in case more than one coding sequence is used in the expression cassette. Typically, each coding sequence found in the expression cassette is operatively linked to at least two regulatory sequences, i.e., a promoter and a polyA sequence.

Any promoter that facilitates a sufficiently high rate of expression can be used in the expression cassette. The promoter may be constitutive or inducible. See, e.g., Resendez et al., 1988, Mol. Cell Biol. 8:4579-4584; Chang et al., 1987, Proc. Natl. Acad. Sci. USA 84:680-684, which describe inducible promoters. The choice of the promoter depends on what cell type is used in the device of the invention, on the species of animal in which the device is to be used, on the location (e.g., tissue or organ, for example the central nervous system) in the animal in which the device is to be used, the desired level of expression, the types and quantities of transcription factors available (i.e., in the cells of the device of the invention as a result of the cell type and the location in the recipient animal), etc. See, e.g., Gossen et al., 1995, Science 268:1766-1769; Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. USA 89:5547-5551 and U.S. Pat. Nos. 5,851,984; 5,849,997; 5,827,687; 5,811,260; 5,789,215; 5,665,578; 5,512,483; 5,302,517; 4,959,313; 4,935,352, which describe promoter sequences useful for the device of the present invention.

Further examples of promoter sequences and elements are, without in any way limiting the present invention, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Nati. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the .beta.-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Omitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Another regulatory element used in the expression cassette is a polyA sequence (or polyA signal). The polyA sequence used should be capable of efficiently inducing polyadenylation of a transcript specific for the coding sequence to which the polyA sequence is operatively linked. See U.S. Pat. Nos. 5,861,290; 5,851,984; 5,840,525 and 5,627,033, which discuss polyA sequences.

In another embodiment, the expression cassette used in the device of the present invention contains an enhancer element, a 5' or 3' untranslated sequence (or region), an intron or a sequence that regulates RNA stability or a combination of more than one of these elements. Any sequence that falls into any of these categories can be used in the device of the present invention. See U.S. Pat. Nos. 5,861,290; 5,851,984; 5,840,525; 5,681,744 and 5,627,033, which discuss these regulatory elements. 5' untranslated sequence refers to the sequence of an mRNA molecule between the transcription initiation site and the translation initiation site. 3' untranslated sequence refers to the sequence of an mRNA molecule between the translation termination site and the polyA tail.

5.4. Selectable Markers

The cells used in the device of the present invention are transfected with the exogenous polynucleotide. The cells that take up the exogenous polynucleotide, typically through integration into their genome, are selected for following transfection. A selectable marker may be included in the exogenous polynucleotide that allows a cell that has the marker to be isolated from cells that do not have the marker. Whether a selectable marker is necessary to prepare the cells used in the device of the present invention, depends on the particular method by which the exogenous polynucleotide is introduced (i.e., transfected) into the cells. For example, if the exogenous polynucleotide is introduced into the cells via microinjection, a selectable marker is much less needed than if electroporation is used. For example, the marker may enable a cell to grow under selective conditions, i.e. conditions under which the cell could not grow if it did not have the marker (e.g., the neomycin resistance gene and the hypoxanthine phosphoribosyltransferase gene). A marker may also provide any other means by which to identify the cell which took up the exogenous polynucleotide (e.g., by staining the cells that took up the exogenous polynucleotide). See, e.g., U.S. Pat. Nos. 5,851,984 and 5,789,215, which describe selectable markers.

5.5. Cells

Any eukaryotic cell type can be used in the device of the present invention, for example fibroblasts, glial cells, keratinocytes, hepatocytes, ependymal cells, bone marrow cells, hippocampal cells, stem cells, embryonic stem cells, hematopoietic stem cells, olfactory mucosa cells, adrenal cells, leukocytes, lymphocytes, chromaffin cells, neurons, cells of the immune system, macrophages, Schwann cells, oligodendrocytes, astrocytes, germline cells, somatic cells, epithelial cells, endothelial cells, adrenal medulla cells, osteoblasts, osteoclasts, myoblasts, pancreatic cells (e.g., of the islets of Langerhans), or a mixtures of more than one of the above, etc. The cells used in the device of the present invention may be cells derived from a mammalian animal, a rodent, a farm animal, a mouse, a rat, a sheep, a dog, a cat, a cow, a pig, a bird, a fish, and most preferably a human. The cells should have the ability to proliferate when grown in culture so that the exogenous polynucleotide can amplify. Following transfection with the exogenous polynucleotide and selection for cells that took up the exogenous polynucleotide, the cells, in a preferred embodiment, should be able to establish a cell line that can be grown, stored, re-grown, etc., for extended periods of time. See, e.g., U.S. Pat. No. 5,814,618, which describes cells useful for the device of the present invention.

When cells are used that are very proliferative, it may be desirably to reduce the rate of proliferation of those cells before they are used in the device of the present invention. See, e.g., U.S. Pat. Nos. 5,853,717; 5,843,431; 5,840,576; 5,833,979 and 5,795,790, which describe methods to control proliferation of cells prior to encapsulation.

Cells used in the device of the present invention are grown, processed and prepared for encapsulation in any way known in the art. See, e.g., U.S. Pat. No. 5,650,148, which describes methods to prepare cells for encapsulation.

5.5.1. Transfection

The exogenous polynucleotide used in the device of the present invention is transfected into eukaryotic cells by any means known in the art. For example, electroporation, calciumphosphate coprecipitation, microinjection, lipofection, etc. can be used. See, e.g., U.S. Pat. Nos. 5,814,618 and 5,789,215, which describe transfection methods.

5.6. Capsules

The cells used in the device of the present invention are encapsulated before they are introduced into an animal. Encapsulation of the cells is designed to prevent an immune reaction to the cells in the host animal (e.g., a patient). For general background, see, e.g., Aebischer et al., 1994, Experimental Neurology 126:151-158; Tresco et al., 1992, Cell Transplantation 1:255-264; Aebischer et al., 1991, Experimental Neurology 111:269-275 and Winn et al., 1991, Experimental Neurology 113:322-329.

Depending on the particular design of the capsule used in the device of the invention, molecules up to a maximum size (i.e., in kilodalton weight ("kD")) will be able to move into and out of the capsule, i.e., molecular cut-off weight. The molecular cut-off weights useful for the device of the present invention is 1 kD, in another aspect it is 2 kD, in another aspect it is 5 kD, in another aspect it is 10 kD, in another aspect it is 25 kD, in another aspect it is 50 kD, in another aspect it is 100 kD and in yet another aspect it is 200 kD, or more. The material of the capsule should be such that it does not lead to any substantial adverse reactions in the host animal and it should allow the encapsulated cells to survive. In addition, the capsule should allow small molecules to move into and out of the interior of the capsule. In particular, if the cells inside the capsule generate a molecule that is designed to be a drug for the host animal, that molecule should be able to leave the capsule. Also, precursor molecules that are required by the cell to generate the drug molecule should be able to move into the capsule.

In a preferred embodiment, the capsule used in the device of the present invention makes it possible to use cells in the device that are derived from an animal different than the host animal. In another preferred embodiment, the cells are from a different species than the host (xenograft). In a further preferred embodiment, the capsule prevents a reaction of the immune system of the host to the cells and molecules from the cells so that, in a preferred aspect, no immunosuppressants are needed.

In another preferred embodiment, the capsule used in the device of the present invention has two layers, e.g., an inner sphere and an outer layer. In a preferred aspect, substantially all cells of the device are located in the inner layer (or sphere). In another aspect, a device of the present invention contains from about 40 to about 1,000 eukaryotic cells, more preferably from about 60 to about 500, more preferably from about 80 to about 250 and most preferably from about 80 to about 120 eukaryotic cells. However, capsules designed to house substantially larger numbers of cells can also be used in the device of the present invention, for example more than 1,000 cells, or more than 10,000 cells, or more than 50,000 cells, or more than 100,000 cells, or more than 500,000 cells, see, e.g., U.S. Pat. No. 5,800,828, which describes capsules that can house large numbers of cells.

Encapsulation methods and materials useful for the device of the present invention are described in, e.g., U.S. Pat. Nos. 5,853,385; 5,837,234; 5,834,001; 5,800,829; 5,800,828; 5,798,113; 5,762,959; 5,750,103; 5,676,943; 5,656,481; 5,653,975; 5,650,148; 5,639,275; 5,550,050 and 5,283,187.

5.7. Administration

The device of the present invention is transferred to a site in a host animal where the metabolic activity that is provided by the device is needed. In cases where the device cannot be transplanted to the most desirable location, it can be placed in a location that is sufficiently close so that the molecule that is provided by the device will be available at the desired site.

The device may be used in any organ or tissue of the recipient animal as the device is small and immunologically tolerable. Tissues in which the device of the invention may be located are connective tissue, muscle tissue, nerve tissue, epithelial tissue, etc. Organs in which the device of the invention may be located are the central nervous system, the spinal cord, the lumbar space, the peripheral nervous system, the pancreas, the liver, the kidneys, the lungs, the heart, the peritoneal cavity, the skeletal muscles, the organ muscles, the bones, cartilage, etc.

See, e.g., U.S. Pat. Nos. 5,800,828; 5,750,103; 5,650,148 and 5,550,050, which describe methods to transfer encapsulated cells into an animal.

5.8. Dosage

The device of the present invention will generally be used in an amount effective to achieve the intended purpose. For use to deliver a molecule lacking in a disease condition to an animal, e.g., a human (patient), the device of the present invention, in a pharmaceutically acceptable preparation thereof, is administered or applied in a therapeutically effective amount. By therapeutically effective amount is meant an amount effective to ameliorate or prevent the symptoms, or prolong the survival of, the animal being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art.

A therapeutically effective dose can be estimated initially from in vitro assays using the molecule delivered by the device of the present invention. In an animal model, a dose can be formulated to achieve a circulating concentration range that includes the IC₅₀ as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

Dosage amount may be adjusted individually to provide plasma levels of a therapeutically desirable molecule provided to the recipient animal by the device of the invention which are sufficient to maintain therapeutic effect.

The amount of the device of the invention administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. Moreover, the specific design of the device of the invention will also determine what is a proper dosage.

The therapy may be repeated intermittently while symptoms are detectable or even when they are not detectable. The therapy may be provided alone or in combination with other drugs.

5.8.1. Toxicity

Preferably, a therapeutically effective dose of a device described herein will provide therapeutic benefit without causing substantial toxicity.

Toxicity of a device described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD₅₀ (the dose lethal to 50% of the population) or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. Devices which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of a device described herein lies preferably within a range of concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending upon the particular design of the device and the location in the recipient animal where the device is utilized. The exact formulation, route and location of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p.1).

Claim 1 of 21 Claims

What is claimed is:

1. An encapsulated population of cells comprising

(a) a eukaryotic cell;

(b) an exogenous polynucleotide comprising an expression cassette and an amplification-promoting sequence, said eukaryotic cell comprising said polynucleotide, wherein said expression cassette is stably expressed and comprises a nucleic acid encoding an insulin, an insulin precursor polypeptide, a serotonin, a neurotropic factor, a neurotrophic factor, a tyrosine hydroxylase, a tryptophan hydroxylase, an enkephalin, an enkephalin precursor polypeptide, an enkephalinase, a melanoma inhibitory activity and parathyroid hormone and wherein said amplification-promoting sequence comprises a polynucleotide that is capable of hybridizing under highly stringent conditions to a polynucleotide that is complementary to SEQ ID NO:1 or SEQ ID NO:2; and

(c) a capsule, said eukaryotic cell being located in the interior of said capsule.

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