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  Pharmaceutical Patents  

 

Title:  Populations of expanded and re-differentiated adult islet beta cells capable of producing insulin and methods of generating same
United States Patent: 
8,039,254
Issued: 
October 18, 2011

Inventors:
 Efrat; Shimon (Zikhron-Yaakov, IL)
Assignee:
  Ramot at Tel-Aviv University Ltd. (Tel-Aviv, IL)
Appl. No.: 
11/791,170
Filed:
 November 21, 2005
PCT Filed: 
November 21, 2005
PCT No.:
 PCT/IL2005/001231
371(c)(1),(2),(4) Date:
 May 21, 2007
PCT Pub. No.:
 WO2006/054305
PCT Pub. Date:
 May 26, 2006


 

Pharm Bus Intell & Healthcare Studies


Abstract

A population of expanded adult islet beta cells is provided as well as a population of expanded and redifferentiated adult islet beta cells. Methods of generation of the populations of cells are provided.

Description of the Invention

SUMMARY OF THE INVENTION

According to the present invention there is provided a population of ex vivo expanded adult islet beta cells being propagatable ex-vivo for at least sixteen passages while demonstrating continuous expression of islet markers.

According to another aspect of the present invention there is provided a population of ex vivo expanded and re-differentiated adult islet beta cells, wherein an insulin content of the expanded and re-differentiated cells is at least 5% of total cellular protein following glucose stimulation.

According to yet another aspect of the present invention there is provided a method of ex-vivo expanding adult islet beta cells comprising incubating adult islet beta cells in a medium comprising CMRL-1066, thereby ex-vivo expanding the adult islet beta cells.

According to still another aspect of the present invention there is provided a method of ex-vivo expanding and re-differentiating adult islet beta cells comprising incubating adult islet beta cells in a medium comprising CMRL-1066, thereby obtaining expanded adult islet beta cells and providing the expanded adult islet beta cells with at least one beta cell differentiation promoting agent, thereby expanding and re-differentiating adult islet beta cells.

According to an additional aspect of the present invention there is provided a method of ex-vivo increasing insulin content in adult islet beta cells comprising providing a quantity of betacellulin sufficient to increase insulin content in the adult islet beta cells.

According to yet an additional aspect of the present invention there is provided a method of treating diabetes in a subject, comprising transplanting a therapeutically effective amount of a population of ex-vivo expanded and re-differentiated adult islet beta cells, whose insulin content is at least 5% of total cellular protein following glucose stimulation, into the subject, thereby treating diabetes.

According to still an additional aspect of the present invention there is provided a use of the population of ex-vivo expanded and re-differentiated adult islet beta cells, whose insulin content is at least 5% of total cellular protein following glucose stimulation, to treat diabetes in a subject.

According to a further aspect of the present invention there is provided a medium for expanding adult islet beta cells comprising medical grade CMRL-1066.

According to yet a further aspect of the present invention there is provided a pharmaceutical composition comprising as an active ingredient the population of ex-vivo expanded and re-differentiated adult islet beta cells whose insulin content is at least 5% of total cellular protein following glucose stimulation, and a pharmaceutically acceptable carrier.

According to further features in preferred embodiments of the invention described below, the adult islet beta cells are trypsinized

According to still further features in the described preferred embodiments the method of ex-vivo increasing insulin content in adult islet beta cells further comprises providing the adult islet beta cells at least one beta cell differentiation promoting agent.

According to still further features in the described preferred embodiments the islet markers comprise beta cell markers.

According to still further features in the described preferred embodiments the beta cell markers comprise PC1/3 and PC2.

According to still further features in the described preferred embodiments the islet markers are selected from the group consisting of Isl-1, somatostatin and pancreatic polypeptide.

According to still further features in the described preferred embodiments the ex-vivo expanded adult islet beta cells are characterized by a replication rate of seven days.

According to still further features in the described preferred embodiments the ex-vivo expanded and re-differentiated adult islet beta cells are glucose responsive.

According to still further features in the described preferred embodiments the medium comprises serum.

According to still further features in the described preferred embodiments the serum comprises fetal calf serum or fetal bovine serum.

According to still further features in the described preferred embodiments the medium further comprises glucose.

According to still further features in the described preferred embodiments a concentration of glucose is 5.6 mM.

According to still further features in the described preferred embodiments the medium comprises antibiotics.

According to still further features in the described preferred embodiments providing the adult islet beta cells at least one beta cell differentiation promoting agent comprises expressing in the adult islet beta cells the at least one at least one beta cell differentiation promoting agent.

According to still further features in the described preferred embodiments providing the adult beta cells at least one beta cell differentiation promoting agent comprises incubating the adult islet beta cells in a medium comprising the at least one beta cell differentiation promoting agent.

According to still further features in the described preferred embodiments providing the adult islet beta cells an amount of betacellulin sufficient to increase insulin content comprises expressing in the adult islet beta cells betacellulin.

According to still further features in the described preferred embodiments providing the adult islet beta cells an amount of betacellulin sufficient to increase insulin content comprises incubating the adult islet beta cells in a medium comprising betacellulin.

According to still further features in the described preferred embodiments the quantity of betacellulin sufficient to increase insulin content is selected from a range between 0.5 and 8 nM.

According to still further features in the described preferred embodiments the medium is CMRL-1066.

According to still further features in the described preferred embodiments medium further comprises glucose.

According to still further features in the described preferred embodiments the medium further comprises antibiotics.

According to still further features in the described preferred embodiments the population of ex-vivo expanded and re-differentiated adult islet beta cells is genetically modified to express a pharmaceutical agent.

According to still further features in the described preferred embodiments the pharmaceutical agent reduces immune mediated rejection.

According to still further features in the described preferred embodiments the insulin content is equivalent to the insulin content in normal adult islet beta cells.

According to still further features in the described preferred embodiments the at least one beta cell differentiation promoting agent is a transcription factor.

According to still further features in the described preferred embodiments the transcription factor is selected from the group consisting of NeuroD, Ngn3, Pax6, Pax4, NRx2.2, NRx6.1, Pdx-1 and Isl-1.

According to still further features in the described preferred embodiments the transcription factor is Ngn3.

According to still further features in the described preferred embodiments the at least one beta cell differentiation promoting agent is betacellulin.

The present invention successfully addresses the shortcomings of the presently known configurations by providing conditions for expanding adult human islet cells, as well as methods for their redifferentiation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of an expanded and re-differentiated population of adult beta cells capable of both storing insulin in physiological amounts and secreting insulin in response to glucose. The present invention can be used in cell replacement therapy in the treatment of insulin dependant diabetes.

The principles and operation of the expanded and re-differentiated isolated population of adult beta cells according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Type I diabetes is caused by the autoimmune destruction of the pancreatic islet insulin-producing beta cells. Insulin administration does not prevent the long-term complications of the disease, since the optimal insulin dosage is difficult to adjust. Replacement of the damaged cells with regulated insulin-producing cells is considered the ultimate cure for type 1 diabetes. Pancreas transplantation has been successful but is severely limited by the shortage of donors. With the development of new islet isolation and immunosuppression procedures, significant success has been reported using islets from 2-3 donors per recipient (Shapiro A M, Lakey J R, Ryan E A et al. New Engl J Med 2000; 343:230-238). This progress underscores the urgent need for developing alternatives to human pancreas donors, namely abundant sources of cultured human .beta. cells for transplantation.

While reducing the present invention to practice, the present inventors have uncovered a novel approach for ex-vivo expansion of isolated adult islet beta cells while still maintaining islet marker expression. The present inventors have also uncovered novel conditions for increasing insulin content in cells (i.e., re-differentiating cells) propagated according to the teachings of the present invention. The present invention exploits these finding to provide a viable source of functioning beta cells for transplantation into diabetic patients.

As is illustrated hereinbelow and in the Examples section which follows the present inventor has uncovered that isolated adult islet beta cells may be propagated in CMRL-1066 for at least sixteen passages, without a noticeable change in cell replication rate and without detectable apoptosis representing an expansion of over 65,000-fold. During propagation, the isolated beta cells undergo de-differentiation (although not complete, as the expanded cells still express a set of islet markers). Dedifferentiation may be measured by a decrease in transcription of key genes expressed in normal, quiescent beta cells, as illustrated in FIGS. 4A-F (see Original Patent). The present inventor has also uncovered that the expanded cell population of the present invention may be re-differentiated by addition of betacellulin and/or Ngn-3. As illustrated in Example 2, the re-differentiated cells are capable of storing physiological concentrations of insulin and secreting therapeutic quantities of insulin in response to glucose.

The present invention overcomes prior art limitations in various aspects. Ex-vivo adult and fetal human islet cells cultured according to prior art teachings have been shown to proliferate at the most for 10-15 population doublings, after which they undergo senescence (Halvorsen T L, et al., J Endocrinol 2000; 166:103-109). The expanded adult islet beta cells of the present invention are propagatable for at least sixteen passages.

Both committed and non-committed cells have been shown to differentiate into insulin producing cells. In sharp contrast to the present invention, these cells are not capable of secreting therapeutic concentrations of insulin in response to glucose.

U.S. Pat. Appl. No. 20040132679 teaches administration of betacellulin in conjunction with islet cell differentiation transcription factors for the treatment of type I Diabetes. In sharp contrast to the present invention, U.S. Pat. Appl. No. 20040132679 does not teach differentiation of an expanded population of adult islet beta cells. Furthermore, when administered alone, betacellulin had no effect on the serum glucose of STZ induced diabetic mice, indicating that betacellulin alone was not able to fully differentiate the hepatic cells.

Thus, according to one aspect of the present invention there is provided a method of ex-vivo expanding adult islet beta cells comprising incubating isolated adult islet beta cells in a medium comprising CMRL-1066, thereby ex-vivo expanding the adult islet beta cells.

As used herein the phrase "ex-vivo" refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube).

As used herein, the phrase "adult islet beta cells" refers to post-natal (e.g., non-embryonic) pancreatic islet endocrine cells which are capable of secreting insulin in response to elevated glucose concentrations and express typical beta cell markers. Examples of beta cell markers include, but are not limited to, insulin, pdx, Hnf3.beta., PC1/3, Beta2, NRx2.2, GLUT2 and PC2.

The isolated adult islet beta cells of this aspect of the present invention may be of homogeneous or heterogeneous nature.

Thus, for example, the adult islet beta cells of this aspect of the present invention may be comprised in isolated pancreatic islets. Islet cells may be comprised of the following: 1) beta cells that produce insulin; 2) alpha cells that produce glucagon; 3) delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide. The polypeptide hormones (insulin, glucagon, somatostatin and pancreatic polypeptide) inside these cells are stored in secretary vesicles in the form of secretory granules.

Methods of isolating islets are well known in the art. For example, islets may be isolated from pancreatic tissue using collagenase and ficoll gradients. An exemplary method is described in Example 1 herein below.

Preferably the adult islet beta cells of the present invention are dispersed into a single cell suspension--e.g. by the addition of trypsin or by trituration.

The adult islet beta cells may be further isolated being substantially free from other substances (e.g., other cells, proteins, nucleic acids, etc.) that are present in its in-vivo environment e.g. by FACs sorting.

The adult islet beta cells may be obtained from any autologous or non-autologous (i.e., allogeneic or xenogeneic) mammalian donor. For example, cells may be isolated from a human cadaver.

As used herein, the term "expanding" refers to increasing the number and overall mass of adult islet beta cells of the present invention by the process of cell division, rather than simply enlarging by hypertrophy. As described in Example 1 herein below, adult islet beta cells may be expanded by passaging the cells every seven days and refeeding twice a week. According to the teachings of the present invention the adult islet beta cells may be expanded 65,000 fold without any detectable apoptosis.

As used herein, the term "CMRL 1066" refers to the serum free medium, originally developed by Connaught Medical Research Laboratories for the culture of L cells, and includes any other derivations thereof provided that the basic function of CMRL is preserved. CMRL-1060 medium is commercially available in either liquid or powder form from companies including Gibco BRL, Grand Island, N.Y., catalogue number 11530-037; Cell and Molecular Technologies, Phillipsburg N.J.; Biofluids Inc, Rockville, Md.; Bioreclamation Inc. East Meadow, N.Y.; United States Biological, Swampscott, Mass.; Sigma Chemical Company, St. Louis, Mo.; Cellgro/Mediatech, Herndon, Va. and Life technologies, Rockville Md.

Preferably the CMRL is of medical grade purity. Thus, there is provided a medium for expanding adult islet beta cells of medical grade purity. As used herein the phrase "medical grade purity" refers to both the constituents of CMRL and the final product being of medical grade purity (i.e., safe for administration). The CMRL medium of the present invention may further comprise supplementary constituents which may improve growth and/or viability of the adult islet beta cells. These include, but are not limited to, growth factors (e.g. hepatocyte growth factor, nerve growth factor and/or epidermal growth factor) serum (e.g. fetal calf serum or fetal bovine serum), glucose (e.g. 5.6 mM) and antibiotics. Exemplary antibiotics and their concentrations are described in the Examples section herein below.

Preferably, the adult islet beta cells are propagated as anchorage-dependent cells by attaching to a solid substrate (i.e., a monolayer type of cell growth). According to a preferred embodiment the adult islet beta cells may be in CMRL 1066 medium at 37.degree. C. with 5% CO.sub.2.

The adult islet beta cells generated according to the above teachings are propagatable ex-vivo for at least sixteen passages while demonstrating continuous expression of islet markers.

According to this aspect of the present invention, the term "continuous expression of islet markers" refers to a detectable mRNA/protein expression of islet markers throughout each round of cell division. Methods of detecting mRNA/protein expression are well known in the art and include but are not limited to Northern, RT-PCR, oligonucleotide microarray, Western, RIA, Elisa, FACS and immunohistochemical analysis.

The phrase "islet markers" refers to at least one mRNA and/or protein which is specifically expressed in the pancreatic islet. Preferably, at least one of the islet markers is a beta cell marker. As shown in the Examples section which follows, the adult islet beta cells expanded according to the teachings of the present invention were shown to express two beta cell markers PC1/3 and PC2. In addition, the expanded adult islet beta cells expressed somatostatin, pancreatic polypeptide and Isl-1.

The expanded adult islet beta cells of this aspect of the present invention do not express all typical beta cell markers. Thus as illustrated in FIGS. 3A-B (see Original Patent), following expansion the adult islet beta cells do not express insulin, pd-x, neuro D and NRx2.2 and may be referred to as being in a state of de-differentiation.

As mentioned hereinabove, while further reducing the present invention to practice, the present inventor uncovered, a method of re-differentiating the expanded adult islet beta cells of the present invention. The method is effected by providing at least one beta cell differentiation promoting agent.

As used herein the term "re-differentiating" refers to the altering of a cell such that it passes from one of a less defined function to one of a more defined function (may also be referred to as more differentiated). For example, the defined functions of an adult beta cell include storing insulin and secreting insulin in response to glucose. Re-differentiation of the expanded adult islet beta cells of the present invention may include such processes as increasing beta cell insulin content, increasing sensitivity to glucose and/or increasing secretory apparatus. Methods of increasing beta cell insulin content may include increasing insulin transcription and/or post transcriptional control and/or increasing translation and/or post-translational control. Methods of increasing beta cell insulin content may also include enhancing insulin storage and/or retarding insulin breakdown. Methods of increasing sensitivity to glucose may include increasing the expression of glucose transporters.

As used herein a "beta cell differentiation promoting agent" refers to a molecule (e.g., a proteninaceous or nucleic molecule) which is able either alone or in combination with other beta cell differentiation promoting agents to re-differentiate expanded adult islet beta cells of the present invention using any of the mechanisms mentioned hereinabove.

Examples of beta cell differentiation promoting agents include but are not limited to Activin A, Atrial Natriuretic Peptide, Betacellulin, Bone Morphogenic Protein (BMP-2), Bone Morphogenic Protein (BMP-4), C natriuretic peptide (CNP), Caerulein, Calcitonin Gene Related Peptide (CGRP-ax), Cholecystokinin (CCK8-amide), Cholecystokinin octapeptide (CCK8-sulfated), Cholera Toxin B Subunit, Corticosterone (Reichstein's substance H), Dexamethasone, DIF-1, Differanisole A, Dimethylsulfoxide (DMSO), EGF, Endothelin 1, Exendin 4, FGF acidic, FGF2, FGF7, FGFb, Gastrin I, Gastrin Releasing Peptide (GRP), Glucagon-Like Peptide 1 (GLP-1), Glucose, Growth Hormone, Hepatocyte Growth Factor (HGF), IGF-1, IGF-2, Insulin, KGF, Lactogen, Laminin, Leu-Enkephalin, Leukemia Inhibitory Factor (LIF), Met-Enkephalin, n Butyric Acid, Nerve Growth Factor (.beta.-NGF), Nicotinamide, n-n-dimethylformamide (DMF), Parathyroid Hormone Related Peptide (Pth II RP), PDGF AA+PDGF BB MIX, PIGF (Placental GF), Progesterone, Prolactin, Putrescine Dihydrochloride Gamma-Irradiated Cell Culture, REG1, Retinoic Acid, Selenium, Selenious Acid, Sonic Hedgehog, Soybean Trypsin Inhibitor, Substance P, Superoxide Dismutase (SOD), TGF-.alpha., TGF-.beta. sRII, TGF-.beta.1, transferrin, Triiodothyronine (T3), Trolox, Vasoactive Intestinal Peptide (VIP), VEGF, Vitamin A and Vitamin E.

A beta cell differentiation promoting agent may also be a transcription factor. The term "beta cell differentiation transcription factor" as used herein is defined as any molecule, either a polypeptide or a nucleic acid expressing the polypeptide, which is involved in beta cell differentiation by functioning as a transcription factor. The transcription factor may also participate in additional mechanisms directed to development, metabolism or the like. Examples of beta cell differentiation transcription factor include, but are not limited to, NeuroD (GenBank Accession No. AAA93480), Pax6 (GenBank Accession No. AAK95849), Pax4 (GenBank Accession No. AAD02289), NRx2.2 (GenBank Accession No. AAC83132), NRx6.1 (GenBank Accession No. AAD11962), Isl-1 (GenBank Accession No. NP002193), Pd-x (GenBank Accession No. AAA88820) or Ngn3 (GenBank Accession No. AAK15022) and homologues or orthologues of same.

According to a preferred embodiment of this aspect of the present invention the beta cell differentiation promoting agent is betacellulin (GenBank Accession No. XP172810) or a functional portion thereof e.g. EGF binding domain [Riese D. J. et al., Oncogene. 1996 Jan. 18; 12(2):345-53]. The term may be used to encompass any polypeptide which comprises betacellulin activity i.e. functions as a ligand for an EGF (epidermal growth factor) receptor protein, and participates in the growth and differentiation mechanisms of islet cells in a pancreas. Determination of betacellulin functional portions which may be used in accordance with the present invention may be achieved using assays familiar to those of skill in the art. In one embodiment, the betacellulin polypeptide is a full-length protein, i.e., preprotein or betacellulin precursor, that has not been proteolytically cleaved such as in amino acid sequences as set forth in AAA40511, Q05928 and P35070. Examples of betacellulin homolog gene sequences include GenBank Accession Nos. AAA40511; NP071592; AAM21214; XP124577; NPO31594; NP001720; BAA96731; AAF15401; AAB25452; AAA40511.

The beta cell differentiation promoting agents of the present invention may be synthesized using any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of polypeptides from natural sources, or the chemical synthesis of polypeptides.

For example beta cell differentiation promoting agents may be synthesized using solid phase peptide synthesis procedures that are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, [Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984)]. Synthetic peptides can be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed by amino acid sequencing.

In cases where large amounts of the peptide are desired, they can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al., (1990) Methods in Enzymol. 185:60-89, Brisson et al., (1984) Nature 310:511-514, Takamatsu et al., (1987) EMBO J. 6:307-311, Coruzzi et al., (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al., (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

The beta cell differentiation promoting agents may be synthesized as fusion proteins. These proteins are typically linked at the N- or C-terminus, to all or a portion of a second polypeptide. In the present invention, a fusion protein may comprise a beta cell differentiation transcription factor sequence and/or a betacellulin sequence together with a linking moiety or a reporter (detectable) molecule. The fusion may aid in stabilizing or assisting in the folding of the beta cell differentiation promoting agent. In other examples, the fusion protein employs leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals or transmembrane regions.

Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art. For example, betacellulin is commercially available from BTC; R&D Systems, Minneapolis, Minn.

The beta cell differentiation promoting agents of the present invention may be purified--e.g. by fractionation Following purification, beta cell promoting agents are typically assayed (e.g. by protein assays) to determine whether the purified protein has retained its activity.

Polypeptide agents for promoting beta cell differentiation may be provided to the adult islet beta cells per se. Alternatively, polynucleotides encoding same may be administered to the adult islet beta cells. In this case, the polynucleotide agent is ligated in a nucleic acid construct under the control of a cis-acting regulatory element (e.g. promoter) capable of directing an expression of the beta cell differentiation promoting agent in the adult islet beta cells in a constitutive or inducible manner.

The nucleic acid construct may be introduced into the expanded cells of the present invention using an appropriate gene delivery vehicle/method (transfection, transduction, etc.) and an appropriate expression system. Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com). Lipid-based systems may be used for the delivery of these constructs into the expanded adult islet beta cells of the present invention. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)] Recently, it has been shown that Chitosan can be used to deliver nucleic acids to the intestine cells (Chen J. (2004) World J Gastroenterol 10(1):112-116). Other non-lipid based vectors that can be used according to this aspect of the present invention include but are not limited to polylysine and dendrimers.

The expression construct may also be a virus. Examples of viral constructs include but are not limited to adenoviral vectors, retroviral vectors, vaccinia viral vectors, adeno-associated viral vectors, polyoma viral vectors, alphaviral vectors, rhabdoviral vectors, lenti viral vectors and herpesviral vectors.

A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-transcriptional modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably, the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the peptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction site and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.

Preferably the viral dose for infection is at least 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, 10.sup.15 or higher pfu or viral particles.

It will be appreciated that expression of more than one beta cell differentiation promoting agent in the expanded cells of the present invention may be desired. Various construct schemes can be utilized to express more than one beta cell differentiation promoting agent from a single nucleic acid construct.

For example, the two recombinant proteins can be co-transcribed as a polycistronic message from a single promoter sequence of the nucleic acid construct.

To enable co-translation of both beta cell differentiation promoting agents from a single polycistronic message, the first and second polynucleotide segments can be transcriptionally fused via a linker sequence including an internal ribosome entry site (IRES) sequence which enables the translation of the polynucleotide segment downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule including the coding sequences of both the first and the second growth factors will be translated from both the capped 5' end and the internal IRES sequence of the polycistronic RNA molecule to thereby produce both beta cell differentiation promoting agents.

Alternatively, the first and second polynucleotide segments can be translationally fused via a protease recognition site cleavable by a protease expressed by the cell to be transformed with the nucleic acid construct. In this case, a chimeric polypeptide translated will be cleaved by the cell expressed protease to thereby generate both beta cell differentiation promoting agents.

Still alternatively, the nucleic acid construct of the present invention can include two promoter sequences each being for separately expressing both beta cell differentiation promoting agents. These two promoters which may be identical or distinct can be constitutive, tissue specific or regulatable (e.g. inducible) promoters functional in one or more cell types.

The beta cell differentiation promoting agents, either alone or in combination, may be provided to ex-vivo cultured adult islet beta cells by addition to the incubating medium. According to one embodiment betacellulin is provided alone in a quantity that is sufficient to increase insulin content in the adult islet beta cells. The phrase "insulin content" refers to the amount of mature insulin inside an adult beta cell. Measurement of insulin content is well known in the art. An exemplary method is extraction of cellular insulin with 3 M acetic acid as described in the Examples section which follows. The amount of mature insulin extracted from the adult islet beta cells may be determined using an ELISA kit commercially available from Mercodia, Uppsala, Sweden.

Typically, the concentration range of betacellulin that is sufficient to increase insulin content is between 0.5 and 8 nM. Preferably, the ex-vivo cultured adult islet beta cells are differentiated with betacellulin for at least six days.

Any medium may be used to incubate the expanded adult islet beta cells in the presence of the beta cell differentiation promoting agent. According to one embodiment, the medium is CMRL-1066. The medium may also comprise other agents such as glucose, serum and antibiotics.

Following the ex-vivo re-differentiation of the expanded adult islet beta cells of the present invention a population of adult islet beta cells are generated whose insulin content is at least 5% of total cellular protein following glucose stimulation.

As used herein the phrase "glucose stimulation" refers to the addition of glucose at a concentration of 16 mM for thirty minutes. This relieves the islet beta cells of a certain percent of their stored insulin.

Methods of determining total cellular protein are well known in the art (e.g. Bradford assay).

Methods of determining insulin content are provided herein above and in the Examples section which follows.

Preferably the amount of insulin in adult islet beta cell is greater than 5% of total cellular protein. Preferably the amount of insulin in adult islet beta cell is 10% of total cellular protein, even more preferably 15% and even more preferably 20%. Restoration of insulin content to 20% of total cellular protein represents a level in the range of that of normal beta cells. As used herein a "normal beta cell" refers to an in-vivo functioning beta cell. The amount of insulin in a normal beta cell may be calculated using the supposition that human pancreata comprise 1-15 grams of insulin, which contains about 10.sup.9 islet cells.

The redifferentiated adult islet beta cells of the present invention are glucose responsive. According to this aspect of the present invention, the phrase "glucose responsive" refers to the ability of the re-differentiated adult islet beta cells of the present invention to secrete insulin in response to glucose. Preferably, the adult islet beta cells secrete at least twice the quantity of insulin in response to 16 mM glucose as they secrete at 0 mM glucose.

The population of adult islet beta cells of the present invention may be further modified (e.g. genetic modification) to express a pharmaceutical agent such as a therapeutic agent, a telomerase gene, an agent that reduces immune mediated rejection or a marker gene. It is contemplated that therapeutic agents such as antimetabolites (e.g., purine analogs, pyrimidine analogs), enzyme inhibitors and peptidomimetics may be generally useful in the present invention. An example of a gene that may reduce immune mediated rejection is the uteroglobin gene. Uteroglobin is a protein expressed during pregnancy that confers immunologic tolerance and prevents inflammatory reactions. Methods of genetically modifying the adult islet beta cells of the present invention are described hereinabove.

Since the adult islet pancreatic cells of the present invention store and secrete insulin, they may be used for treating a disease which is associated with insulin deficiency such as diabetes.

Thus, according to another aspect of the present invention there is provided a method of treating diabetes in a subject, the method comprising transplanting a therapeutically effective amount of the population of ex-vivo expanded and re-differentiated adult islet beta cells of the present invention into the subject, thereby treating diabetes.

As used herein "diabetes" refers to a disease resulting either from an absolute deficiency of insulin (type 1 diabetes) due to a defect in the biosynthesis or production of insulin, or a relative deficiency of insulin in the presence of insulin resistance (type 2 diabetes), i.e., impaired insulin action, in an organism. The diabetic patient thus has absolute or relative insulin deficiency, and displays, among other symptoms and signs, elevated blood glucose concentration, presence of glucose in the urine and excessive discharge of urine.

The phrase "treating" refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition in an individual suffering from, or diagnosed with, the disease, disorder or condition. Those of skill in the art will be aware of various methodologies and assays which can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays which can be used to assess the reduction, remission or regression of a disease, disorder or condition.

As used herein, "transplanting" refers to providing the redifferentiated adult islet beta cells of the present invention, using any suitable route. Typically, beta cell therapy is effected by injection using a catheter into the portal vein of the liver, although other methods of administration are envisaged.

As mentioned hereinabove, the adult islet beta cells of the present invention can be derived from either autologous sources or from allogeneic sources such as human cadavers or donors. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolating, semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu M Z, et al., Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol. Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J. Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 .mu.m. Such microcapsules can be further encapsulated with additional 2-5 .mu.m ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 .mu.m (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE.sup.R), etanercept, TNF.alpha. blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

The adult islet beta cells of the present invention may be transplanted to a human subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the adult islet beta cells of the present invention accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (insulin producing cells) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., diabetes) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated from animal models (e.g. STZ diabetic mice) to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in experimental animals. The data obtained from these animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route 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).

Dosage amount and interval may be adjusted individually to provide cell numbers sufficient to induce normoglycemia (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.
 

Claim 1 of 2 Claims

1. A population of dedifferentiated adult islet cells generated by incubating isolated adult islet beta cells in a medium comprising CMRL-1066 and glucose, wherein a majority of said dedifferentiated adult islet cells express PC1/3 and do not express insulin.

 

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