WO2009067644A1 - Compositions, systems and methods for obtaining and expanding insulin-producing cells - Google Patents

Abstract

The invention encompasses compositions, systems, and methods relating to miRNAs and their target genes that are involved in biogenesis, and/or replication, and/or regeneration of insulin-producing cells miRNAs (mir-7, mir-141, mir-127, mir-382 mir-299-5p and mir221) were identified that are differentially expressed in pancreatic islets, mir-7 being the most abundant In the developing mouse pancreas, expression of mir-7 precedes the expansion of insulin-producing cells. The SPATA2 gene was predicted and validated as a mir-7 target, and the expression of SPATA2 transcripts inversely correlated with mir-7 during mouse pancreatic development. The compositions and methods descnbed herein should prove useful for restoring J3-cell function, inducing differentiation of noninsulin- producing cells into insulin-producing cells, and/or inducing proliferation of insulin producing cells, and inducing regeneration of insulin- producing cells in a subject.

Description

COMPOSITIONS, SYSTEMS AND METHODS FOR OBTAINING AND EXPANDING INSULIN-PRODUCING CELLS

FIELD OF THE INVENTION

[0001] The invention relates generally to the fields of developmental biology, molecular biology, regenerative therapies, and endocrinology. More particularly, the invention relates to modulating the expression of miRNAs and their target genes for inducing biogenesis, proliferation, and regeneration of insulin-producing cells.

BACKGROUND

[0002] Islets of Langerhans are specialized endocrine cell clusters that represent 1 -2% of pancreatic tissue. Four different cell types compose each cluster, the glucagon-producing α- cells and insulin-producing β-cells constituting the majority of these cells. The highly regulated production and secretion of endocrine hormones by islet cells contribute to glucose homeostasis. The reduction of insulin-producing cell function and/or mass results in impaired glucose metabolism and diabetes in humans. Insulin deficiency is accompanied by hyperglycemia, a condition in which an excessive amount of glucose circulates in the blood plasma. Patients with diabetes require the introduction of exogenous insulin treatment, which improves prognosis but cannot restore physiological glycemic control throughout the day. The result of unstable glycemic control is the progression of serious diabetic complications that are associated with high morbidity and mortality rates as well as high medical costs worldwide.

[0003] Restoration of β-cell function, which would provide physiologic glycemic metabolic control, is a highly desirable goal for the treatment of patients with diabetes. Despite certain developments, no methodology exists for restoring β-cell function or inducing insulin-producing cell differentiation and proliferation/regeneration in a patient at levels sufficient to provide physiologic glycemic metabolic control. There is a need for methods of restoring β-cell function, inducing non-insulin-producing cells to differentiate into insulin- producing cells, inducing β-cell proliferation, and inducing β-cell regeneration (e.g., in a patient). SUMMARY

[0004] The invention relates to the development of compositions, systems, and methods involving non-coding microRNAs (miRNAs) for inducing differentiation of non-insulin- producing cells into insulin-producing cells, inducing proliferation of insulin-producing cells, inducing regeneration of insulin-producing cells, restoring β-cell function, and screening cells that can differentiate into insulin-producing cells. miRNAs are non-coding gene products that regulate gene expression through specific binding to target mRNAs. Cell-specific patterns of miRNAs are thought to be necessary for the acquisition/maintenance of specialized cell phenotypes such as those of pancreatic exocrine and endocrine cells. Each miRNA can have multiple targets, including, but not limited to, messenger RNAs alone or in combination with other miRNAs, thus miRNAs are capable of regulating complex regulatory networks, including glucose metabolism.

[0005] In the experiments described below, a subset of miRNAs (mir-7, mir-127, mir-382, mir-141, mir-299-5p and mir-221) was identified that are differentially expressed in pancreatic islets, mir-7 being the most abundant. In the developing mouse pancreas, expression of mir-7 precedes the expansion of insulin-producing cells. Spermatogenesis associated 2 gene (SPATA2), a gene originally described in spermatogenesis, was predicted and validated as a mir-7 target. The expression of SPATA2 transcripts inversely correlated with mir-7 during mouse pancreatic development. In vivo loss of function studies showed that inhibition of mir-7 activity in the fetal pancreas resulted in inhibition of formation of insulin in the fetal pancreas. The results described below show that mir-7 is a marker of differentiated endocrine cells and plays a role in islet biogenesis. The compositions, systems, and methods described herein should prove useful for restoring β-cell function and inducing non-insulin-producing cells to differentiate into insulin-producing cells, and/or promoting proliferation and regeneration of insulin-producing cells in vitro and in a mammalian subject. [0006] Accordingly, described herein is a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of a miRNA that modulates expression of at least one miRNA target gene in a cell for inducing at least one of: differentiation of non-insulin-producing cells into insulin-producing cells, proliferation of insulin-producing cells, and regeneration of insulin-producing cells. The at least one miRNA target gene can be one of SPATA2, Zeb2, SOX4, and CDKNBlB, for example. The miRNA can be mir-7 miRNA and can be within a vector. [0007] Also described herein is a method of inducing insulin-producing cell proliferation in vitro. The method includes the steps of: providing a composition including a pharmaceutically acceptable carrier and an effective amount of a miRNA that modulates expression of at least one miRNA target gene in a cell for inducing at least one of: differentiation of non-insulin-producing cells into insulin-producing cells, proliferation of insulin-producing cells, and regeneration of insulin-producing cells; contacting a plurality of insulin-producing cells with the composition; and culturing the plurality of insulin-producing cells under conditions in which they proliferate. Any suitable conditions in which insulin- producing cells can be cultured to proliferate can be used and are known in the art. The at least one miRNA target gene can be one of SPATA2, Zeb2, SOX4, and CDKNBlB, for example. The miRNA can be mir-7 miRNA, and can be included within a vector. [0008] Also described herein is a method of inducing differentiation of non-insulin- producing cells into insulin-producing cells in vitro. The method includes the steps of: providing a composition including a pharmaceutically acceptable carrier and an effective amount of a miRNA that modulates expression of at least one miRNA target gene in a cell for inducing at least one of: differentiation of non-insulin-producing cells into insulin-producing cells, proliferation of insulin-producing cells, and regeneration of insulin-producing cells; contacting a plurality of non-insulin-producing cells with the composition; and culturing the plurality of non-insulin-producing cells under conditions in which they differentiate into insulin-producing cells. Any suitable conditions in which non-insulin-producing cells can be cultured to differentiate into insulin-producing cells can be used and are known in the art. The at least one miRNA target gene can be one of SPATA2, Zeb2, SOX4, and CDKNBlB, for example. The miRNA can be mir-7 miRNA and can be included within a vector. [0009] Further described herein is a method of modulating insulin expression in a mammal. The method includes the steps of: providing a composition including a therapeutically effective amount of mir-7 miRNA and a pharmaceutically acceptable carrier; and administering the composition to the mammal, wherein administration of the composition to the mammal modulates insulin expression in the mammal. The composition can be administered to the pancreas of the mammal (e.g., by injection). The mir-7 miRNA can be included within a vector. The mir-7 miRNA can further modulate the expression of a target gene such as SPATA2, Zeb2, SOX4, and CDKNBlB, for example. [0010] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0011] As used herein, "treatment" and "treating" are intended to refer to inhibiting, eliminating, ameliorating, diminishing and/or reducing cellular damage and/or symptoms associated with a disease or condition, e.g. diabetes. "Treating" includes, but is not limited to, restoring β cell function, maintaining or promoting glucose homeostasis, etc.. Compositions as described herein may also or alternatively be a prophylactic, i.e., used to partially or completely prevent a disease or condition or symptom thereof.

[0012] Where the terms "patient" and "subject" are used interchangeably in the present specification, they include animals. In one embodiment, the patient is a mammal, and in a preferred embodiment, the patient is human.

[0013] As used herein, "an effective amount" or "a therapeutically effective" amount is intended to refer to the total amount of the active compound of the method that is sufficient to show a meaningful patient benefit. This term is also intended to refer to an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with induced cellular damage.

[0014] As used herein, "insulin producing cells" includes mammalian (e.g., human) cells that synthesize, express, or secrete insulin in a constitutive or inducible manner. One example of an insulin-producing cell is a β cell.

[0015] By the term "non-insulin-producing cells" is meant any mammalian (e.g., human) cells that are not expressing, synthesizing, or secreting insulin in a physiological or in vitro setting that can be transformed into cells that express, synthesize, or secrete insulin.

[0016] miRNA molecules are known in the art (see, for example, Bartel, Cell, 2004, 116,

281-297 for a review on microRNA molecules). The definitions and characterizations of microRNA molecules in the article by Bartel are hereby incorporated by reference. The terms

"miRNA" and "microRNA" are used interchangeably throughout.

[0010] Although compositions, systems, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, systems and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a table showing islet-specific miRNAs selected by significant analysis of microarrays (SAM) of rat islets versus rat acinar miRNA arrays. The expression of miRNAs was examined by micro-arrays containing the rat Sanger mirBase release 8.1 list. SAM analysis with False Discovery Rate (FDR)<5%. Data from six hybridizations was used to determine a signature miRNA list preferentially expressed in islets. The score (d) represents the value of the T-statistic. A higher score means a greater difference between the two groups. Q values represent the chance that the miRNA is a false positive. [0012] FIG. 2 is a pair of graphs showing results from a quantitative real time polymerase chain reaction (qRT-PCR) analysis of miRNAs, evidencing pancreatic miRNAs that are differentially expressed in islets. Values are presented as calculated Relative Quantification (RQ) number to determine the change in expression of rat miRNA transcripts in islets relative to acinar tissue (a) or relative to mir-7 for intraislet comparison (b). Data represent mean ± SD, n=3. Ten miRNAs were selected to validate the microarray analysis. Mir-7, mir-127 and mir-382 constitute a subset of endocrine islet miRNAs that are the most differentially expressed, < 100 fold change, Mir-7 is expressed more than 150-fold compared to acinar tissue and is nine-fold more abundant in islets than mir-382 and mir-127. Conversely, of the islet- specific miRNAs, mir-375 is the most abundantly expressed miRNA in islets (panel b), but the differential expression with acinar tissue is significantly lower than that of mir-7 versus acinar.

[0013] FIG. 3 is a series of graphs showing expression of mir-7, mir-375, insulin and

SPATA2 transcripts during mouse pancreatic development. (1-3) Values are presented as relative quantification (RQ) numbers calculated to determine the change of transcript expression in pancreatic buds corresponding to el3.5, el4.5, el8.5 and neonate stages relative to el 1.5 buds. (4) RQ values of mir-7 and SPATA2 are presented as %. The maximal value in each series equals 100%. Data represent mean ± SD, n=3. Values of P< 0.05 (*) were considered statistically significant. P values were calculated relative to el3.5 RQ values.

[0014] FIG. 4 is a graph illustrating expression of mir-7 and mir-375 during mouse pancreatic development: a comparison between mir-7 and mir-375 transcripts expression in el 1.5, el3.5, el4.5, el8.5 and neonate pancreatic stages. Values are presented as RQ number to determine changes in mir-375 relative to mir-7. [0015] FIG. 5a illustrates the first 3 predictive RNA targets for mir-7 by TargetScan algorithm. SPATA2 has the highest score assigned by this algorithm. The most negative values have the maximal score.

[0016] FIG. 5b illustrates three conserved sites for mir-7 in the 3'UTR of the mouse

SPATA2 gene (NM-006038); two 8mer sites and one 7mer-m8 site. Additionally, one poorly conserved 7mer-m8 site is present at position 1476-1482 (not shown).

[0017] FIG. 6 is a pair of graphs illustrating regulation of mir-7 expression in insulinoma cell line MIN6. (a) mir-7 activity was inhibited by MO. MIN6 cells were cultured 48h with a mixture of lOOμM morpholino (MO) against each mmu-mir-7 and mmu-mir-7-b, the two mature mir-7 miRNAs expressed in mice, (b) Overexpression was achieved by incubation with premir-7 RNA. MIN6 cells were treated with 5nM premir-7 RNA or negative premir RNA control for 32hs. Following incubations in (a) and (b), RNA was isolated, and insulin, glucagon and SPATA2 transcripts were quantified by RT-PCR. Values are presented as RQ calculated to determine changes of transcript expression in cells treated with anti-mir-7 MO or premir-7 RNA relative to negative controls. Data represent mean ± SD, n=3. Normalized fractional cycle number (Ct) at which the PCR product reaches the threshold of detection for all groups was used to perform group comparisons using the paired two-tailed t- test. The calculated p values are: values of P< 0.05 (*) are considered statistically significant. [0018] FIG. 7A is a series of graphs showing the mir-7 expression profile in fetal pancreas. Expression in fetal human pancreas from 8 to 22 wga of gestational age by quantitative PCR of (A) miR-7, insulin, glucagon and somatostatin, (B) miR-7, miR-127, miR-382, miR-375 and (C) miR-7 and NGN3. Data are expressed as means ± SEM.

[0019] FIG. 7B is a series of micrographs showing mir-7 in situ hybridization. In situ hybridization of fetal (9, 10, 11, 12, 14 and 17 wga (w)) and adult human pancreatic tissue using fluorescein-labeled miR-7 LNA probe. Control: 14 wga human pancreatic tissue was hybridized with sense miR-159 LNA oligonucleotide. There is no known target for this probe in the Sanger miRBase. (a) and (b) are higher magnification of areas indicated by arrows. Magnification: 1OX except adult, (a) and (b) which are 2OX.

[0020] FIG. 8 is a series of micrographs showing localization of miR-7 in the developing human pancreas. In situ hybridization of a 14 wga fetal human pancreas using fluorescein-labeled miR-7 LNA probe or DIG-labeled NGN3 probe, counterstained with antibodies against IPFl /PDXl, ISLl, insulin, glucagon and somatostatin, all green except ISLl when together with NGN3. In the merged images in situ hybridization is pseudocolored in red (miR-7) or blue (NGN3). Magnification: 2OX. Inserts in the merged images (a'-f ) are enlarged in the right panels. Scale bars=50μm.

[0021] FIG. 9 is a series of micrographs showing localization of miR-7 in adult human pancreas. In situ hybridization of adult human pancreas using fluorescein-labeled miR-7 LNA probe, counterstained with antibodies against insulin, glucagon, somatostatin, CKl 9 and amylase. In the merged images in situ hybridization is pseudocolored in red. Magnification:

2OX. Scale bars=100μm.

[0022] FIG. 10 is a pair of micrographs showing inhibition of insulin expression by in vivo delivery of MO against miR-7. MO anti-miR-7 (A) or MO irrelevant (B) were intra- heart injected into elθ.5 fetuses. Pancreatic buds were retrieved at day el4.5 and analyzed by immunostaining. Red= glucagon; green = insulin; blue = DAPI staining.

DETAILED DESCRIPTION

[0023] The invention encompasses compositions, systems, and methods relating to miRNAs and their target genes that are involved in biogenesis and/or replication of insulin- producing cells. Expression profile experiments indicated several miRNAs that are differentially expressed in pancreatic islets. Two of these miRNAs, mir-7 and mir-141, were observed among the most differentially expressed miRNAs between acinar tissue and isolated islets of Langerhans, a specialized cluster of endocrine cells formed by four different types of cells (including glucagon-producing α-cells and insulin-producing β-cells). Two other differentially expressed miRNAs, mir-299-5p and mir-221, had an expression profile that varied during pregnancy-induced islet replication. All of mir-7, mir-127, mir-382, mir-141, mir-299-5p and mir-221 have a specific expression profile during mouse pancreatic development. In the developing mouse pancreas, the expression profile of mir-7 strongly correlates with β-cell biogenesis, following the expression of glucagon-producing α-cells. Additionally, similar patterns of expression were observed for mir-141 and mir-221. The expression of mir-299-5p peaks at the beginning of β-cell biogenesis. The required sequence complementarity of the miRNAs with their target mRNAs allows the prediction of possible target mRNAs for each miRNA. Using a bioinformatics platform, potential targets for mir-7, mir-141, mir-299-5p and mir-221 were identified. The SPATA2 gene was identified as a potential target of mir-7; zinc finger E-box binding homeobox 2 (Zeb2) was identified as a potential target of mir-141; SRY (sex determining region Y)-box 4 (SOX4) was identified as a potential target of mir-299-5p; and cyclin-dependent kinase inhibitor IB (p27, Kipl) (CDKNBlB) was identified as a potential target of mir-221. In vivo loss of function studies showed that inhibition of mir-7 activity in the fetal pancreas resulted in inhibition of formation of insulin in the fetal pancreas. The results described below show that mir-7 is a marker of differentiated endocrine cells and plays a role in islet biogenesis. [0024] In addition to those potential target genes described herein (e.g., SPATA2,

Zeb2, SOX4, CDKNBlB), the invention encompasses compositions and methods for regulating the expression of any target gene for inducing insulin-producing cell differentiation and/or proliferation/regeneration, restoring β-cell function, and screening cells that can differentiate into insulin-producing cells. The expression of one or more target genes can be modulated using miRNAs as described herein as well as any other compositions, reagents, or molecules capable of modulating gene expression.

[0025] The below described preferred embodiments illustrate adaptations of these compositions, systems and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

Biological Methods

[0026] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). RT-PCR techniques are also generally known in the art and are described in detail in methodology treatises such as Real Time PCR (Methods in Molecular Biology) by David Sugden, Humana Press, Totowa, NJ, 1^st ed., 2008; and Real Time PCR (BIOS Advanced Methods) by Tevfik Dorak, Taylor and Francis Group, Boca Raton, FL, 1^st ed., 2006; as well as in Tang et al., Nat Protoc. 1(3): 1154- 1159, 2006; and Chen et al., Nucleic Acids Res. 33(20):el79, 2005. These references are herein incorporated by reference.

MicroRNAs

[0027] Described herein are compositions, systems and methods relating to miRNA target genes involved in glycemic metabolic control and miRNAs that modulate target gene expression in a cell (e.g., decrease SPATA2 expression), that induce insulin-producing cell differentiation (i.e., induce non-insulin-producing cells into insulin-producing cells) and/or proliferation/regeneration of insulin-producing cells, and that restore β-cell function in a subject (e.g., mammal). Generally, miRNAs are derived from genomic loci and are produced from specific microRNA genes. Described herein are isolated DNA or RNA molecules that include at least ten contiguous bases having a sequence homologous to that of a pancreatic islet miRNA, such as mir-7, mir-127, mir-382, mir-141, mir-299-5p, or mir-221, and equivalents thereof. In addition to the at least ten contiguous nucleotides of the pancreatic islet miRNA, the isolated DNA or RNA molecule may also have one or more additional nucleotides. There is no upper limit to the additional number of nucleotides. Typically, no more than about 500 nucleotides, and preferably no more than about 300 nucleotides are added to the at least ten contiguous bases of a pancreatic islet miRNA. Any nucleotide can be added. The additional nucleotides can include any base described above. Thus, for example, the additional nucleotides may be any one or more of A, G, C, T, or U.

[0028] In one embodiment, a composition for inducing insulin-producing cell differentiation and/or proliferation and regeneration includes at least one microRNA (e.g., one or more of: mir-7, mir-127, mir-382, mir-141, mir-299-5p and mir-221). The at least one microRNA can be encoded by a vector (e.g, viral vector). In another embodiment, a composition for inducing insulin-producing cell differentiation and/or proliferation and regeneration includes at least one agent that modulates expression of at least one miRNA target gene (e.g., SPATA2, Zeb2, SOX4, and CDKNBlB) in a cell. In addition to those miRNAs (e.g., mir-7, mir-127, mir-382, mir-141, mir-299-5p and mir-221) and those potential target genes (e.g., SPATA2, Zeb2, SOX4, CDKNBlB) described herein, the invention encompasses compositions and methods for regulating the expression of any miRNA and any target gene(s) involved in glycemic metabolic control. [0029] miRNAs are non-coding small RNAs (-19-22 nt) that regulate gene expression by post-transcriptional interference with specific messenger RNAs (mRNA) (Bartel, D. P. Cell 116, 281-97 (2004)). As each miRNA can have multiple target messenger RNAs individually or jointly with other miRNAs, miRNAs are able to control highly complex regulatory network of gene expression (Kim, V.N. & Nam, J.W. Trends Genet 22, 165-73 (2006)). The current number of human miRNA genes listed in the corresponding database is 474. It was suggested that this number will increase up to 1,000, which would amount to almost 3% of the protein-coding genes (Bartel, D.P. Cell 116, 281-97 (2004)). However, a new mammalian miRNA atlas describes 300,000 sequences from 256 small RNA libraries and presents evidence for expression of approximately 400 miRNA genes in each genome (Landgraf, P. et al. Cell 129, 1401-14 (2007)).

[0030] Any suitable miRNAs and their corresponding targets can be used in compositions, systems, and methods of the invention to induce insulin-producing cell differentiation and/or proliferation/regeneration, to restore β-cell function, and to screen cells that can differentiate into insulin-producing cells. In the experiments described herein, mir-7, mir-127, mir-382, mir-183, mir-382, and mir-127 were found to be differentially expressed in pancreatic islets. However, it is envisioned that additional miRNAs may be involved in regulating biogenesis and/or replication of insulin-producing cells. Therefore, the invention encompasses any miRNAs and their corresponding targets that are involved in regulating biogenesis and/or replication of insulin-producing cells. Such additional miRNAs and their targets can be identified and characterized utilizing the methods described herein. [0031] miRNAs as described herein include miRNAs that have been modified (e.g., at a base moiety, sugar moiety) to enhance delivery, stability and/or function. Such modifications include covalent and non-covalent modifications. As an example, miRNAs can be conjugated to groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane. miRNAs as described herein can be modified to have increased nuclease resistance. Such modified moieties are well known in the art, and were reviewed, for example, by Kurreck et al., (Eur. J. Biochem. 270, 1628-1644 (2003)). [0032] A pancreatic islet miRNA (or haipin precursor) as described herein can be inserted into a vector, such as, for example, a recombinant vector. See for example, Chen et al. (Science 2004, 303:83-86). The recombinant vector may be any recombinant vector, such as a plasmid, a cosmid or a phage. Recombinant vectors generally have an origin of replication. The vector may be, for example, a viral vector, such as an adenovirus vector or an adeno-associated virus (AAV) vector. See for example: Ledley 1996, Pharmaceutical Research 13:1595-1614 and Verma et al. Nature 1997, 387:239-242. The vector may further include a selectable marker. Suitable selectable markers include a drug resistance marker, such as tetracycline or gentamycin, or a detectable gene marker, such as β-galactosidase or luciferase.

System for Screening Cells

[0033] One or more miRNAs and/or their target genes as described herein can be used as a screening tool for potential precursors of cells that can be differentiated into insulin- producing cells. Any suitable method or system for screening miRNAs and their targets can be used to identify cells that are capable of producing insulin. In a typical method or system, the expression profile of a set of miRNAs (e.g., 2, 3, 4, 5, etc.) and/or respective target genes (e.g., 2, 3, 4, 5, etc.) is examined in a cell to be screened. Whether or not a particular cell is capable of producing insulin can be determined by the expression of the set of miRNAs and/or their respective genes. Examples of such methods and systems include microarrays, qRT-PCR kits, in situ hybridization kits, and Luminex assays (Invitrogen, Carlsbad, CA). [0034] Any suitable analytical/quantitative methods for determining either microRNAs or their respective targets analyzed as proteins or at the level of messenger RNA (mRNA) can be used and are known. Examples of suitable methods for analysis of microRNAs include: miRNA microarrays, PCR-based microRNA arrays, Luminex-locked nucleic acid (LNA) hybridization arrays, individual quantitative RT-PCR Taqman assays and detection by miRNA in situ hybridization. Examples of suitable methods for mRNA determination include; RNA gene arrays, such as Affymetrix, Agilent or Illumina arrays, individual quantitative RT-PCR Taqman analysis, and Northen analysis. Examples of suitable methods for analysis/determionation of target miRNAs assessed as proteins include: Western analysis, Elisa and Luminex technology and proteomic analysis.

Identifying Targets for Treatment of Diabetes

[0035] Described herein are compositions, systems, and methods for identifying targets for treatment of diabetes. In a typical method of identifying targets for treatment of diabetes, modulation of microRNA expression (e.g., induction of overexpression by gene transfer or inhibition/downregulation by the means of selective inhibitors or MO's) such as mir-7, mir- 127, mir-382, etc., in pancreatic islet cells or in cells that are being differentiated into insulin-producing cells (e.g. D'Amour KA et al. Nat Biotechnol. 2006 24(11):1392-401; Kroon E et al. Nat Biotechnol. 2008 26(4):443-52) is first examined. This analysis may result in differential expression of microRNAs, genes, mRNAs, proteins and intracellular signal transduction pathways that can be assessed using molecular and/or biochemical assays. Such an analysis may be performed in an in vitro and/or in vivo setting to allow for the induction of β cell proliferation/regeneration from stem cell precursors, transdifferentiation of cells, or from existing adult β cells. Subsequent to analyzing microRNA modulation, global gene expression is generally analyzed to identify those genes whose expression was regulated either by inhibition or enhancement of the above-mentioned microRNAs. Global gene expression analysis at the level of mRNA and protein can be achieved as described above (gene microarrays, and/or proteomic analysis). Selective modulation (i.e., via molecular, chemical or physical means) of such targets may lead to the development of reproducible protocols for the restoration of a functional β cell mass in patients with diabetes via cellular therapies or by reprogramming/enhancing β cell self-renewal directly in vivo. Modulating mir-7 Expression and Insulin Expression In Cells

[0036] Described herein are compositions and methods for modulating mir-7 expression and for modulating insulin expression in mammalian cells. These compositions and methods will find use in the treatment of diabetes. For example, in a typical embodiment of a method of modulating mir-7 expression in a mamma suffering from diabetes, a composition including a therapeutically effective amount of an agent that increases or upregulates mir-7 expression or activity is administered to the mammal suffering from diabetes. By upregulating mir-7 expression or activity, insulin production can be increased in the mammal. In one embodiment of a method of modulating mir-7 and insulin expression in cells, a suitable amount (therapeutically effective amount) of mir-7 alone or in combination with one or more of the other pancreatic endocrine miRNAs described herein (e.g. mir-127, mir-382) is introduced into mammalian cells. The pancreatic islet microRNA or haipin precursor can be inserted into a vector, such as, for example, a recombinant vector. Suitable examples of recombinant vectors include plasmids, cosmids and phages. The vector may be, for example, a viral vector, such as an adenovirus vector or an adeno-associated virus (AAV) vector. Any suitable methods for introducing pancreatic endocrines microRNAs into cells can be used, e.g., by microinjection, through a delivery system such as liposomes and charged lipids, dendrimers, biodegradable polymers, polymers of amino acids or sugars, etc. [0037] Similarly, in a typical method of increasing insulin production in a mammal suffering from diabetes, a therapeutically effective amount of an agent that increases or upregulates mir-7 expression or activity or a therapeutically effective amount of mir-7 miRNA is administered to a mammal suffering from diabetes, resulting in increased production of insulin in the mammal.

[0038] Administration of microRNAs to a mammal can be achieved by any suitable methods such as, for example, any oral and systemic enteral or parenteral administration. The targeting of microRNAs to particular pancreatic endocrine cells can be achieved by conjugation of the microRNAs to antibodies recognizing specific receptors expressed on the cell surface. Alternatively, modulation of the expression of miRNA(s) or gene expression downstream of mir-7 may prove an effective approach to attain the induction of critical regenerative pathways to obtain an improved functional β cell mass in patients affected by diabetes.

Methods of Inducing Insulin-Producing Cell Proliferation In Vitro

[0039] Described herein are methods of inducing insulin-producing cell proliferation in vitro. In a typical method, a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of a miRNA that modulates expression of at least one miRNA target gene in a cell for inducing at least one of: differentiation of non-insulin- producing cells into insulin-producing cells, proliferation of insulin-producing cells, and regeneration of insulin-producing cells is first provided. A plurality of insulin-producing cells are contacted with the composition, and the plurality of cells are cultured under conditions in which they proliferate. Any suitable conditions can be used for culturing the insulin-producing cells such that they proliferate (i.e., expand). Methods of proliferating (expanding) insulin-producing cells are known and are described in, for example, Limbert et al, Diabetes Res. Clin. Pract. 79:389-399, 2008; Russ et al, Diabetes 57(6): 1575-1583, 2008; and Parnaud et al., Diabetologica 51(1): page 1432, 2008. In the methods described herein, the at least one miRNA target can be one of SPTA2, Zeb2, SOX4, and CDKNBlB, for example. In a typical embodiment, the miRNA is mir-7 miRNA, and is included within a vector. In some embodiments, the composition can include two or more miRNAs (e.g., mir-7 and mir-127; mir-7 and mir-382; mir-7, mir-127, and mir-382; etc.) that modulate expression of one or more miRNA target genes in a cell. Methods of Inducing Differentiation of Non-Insulin-Producing Cells Into Insulin-Producing

Cells In Vitro

[0040] Described herein are methods of differentiating non-insulin-producing cells into insulin-producing cells in vitro. In a typical method, a composition including a pharmaceutically acceptable carrier and an effective amount of a miRNA that modulates expression of at least one miRNA target gene in a cell for inducing at least one of: differentiation of non-insulin-producing cells into insulin-producing cells, proliferation of insulin-producing cells, and regeneration of insulin-producing cells is first provided. A plurality of non-insulin-producing cells is contacted with the composition, and the plurality of cells are cultured under conditions in which they differentiate into insulin-producing cells. Any suitable conditions can be used for culturing the non-insulin-producing cells such that they differentiate into insulin-producing cells. Methods of culturing non-insulin-producing cells under conditions in which they differentiate into insulin-producing cells are known, and are described in, for example, D'Amour et al., Nat Biotechnol. 24:1392-1401, 2006; Kroon et al, Nat Biotechnolo. 26:443-452, 2008; and U.S. Patent No. 7,056,734. In the methods described herein, the at least one miRNA target can be one of SPTA2, Zeb2, SOX4, and CDKNBlB, for example. In a typical embodiment, the miRNA is mir-7 miRNA, and is included within a vector. In some embodiments, the composition can include two or more miRNAs (e.g., mir-7 and mir-127; mir-7 and mir-382; mir-7, mir-127, and mir-382; etc.) that modulate expression of one or more miRNA target genes in a cell.

Cells and Tissues

[0041] The invention provides compositions including at least one agent (e.g., microRNA) that modulates the expression of a target gene that is involved in β-cell differentiation and/or replication/regeneration and methods of inducing insulin-producing cell differentiation and/or proliferation/regeneration in vitro. Modulating the activity of miRNAs involved in glucose metabolism as described herein alone or in conjunction with manipulation of the expression of their target genes in particular cell types can be utilized to promote the differentiation of non-insulin-producing cells into insulin-producing cells, and/or to promote proliferation/regeneration of insulin-producing cells. Any self-renewable cell type as well as any other primary cell culture or cell line of potential interest can be treated (e.g., induced to proliferate and/or differentiate/transdifferentiate, into an insulin-producing cell) using the compositions and methods described herein. Examples of such cells include, but are not limited to, adult pancreatic tissue, fetal pancreatic tissue, pancreatic stem/progenitor cells, embryonic stem cells, adult islets, neonatal islets, acinar cells, ductal cells, β-cells, placenta-derived stem cells, amniotic fluid-derived stem cells, hepatic oval cells, cord blood cells, mesenchymal stem cells, bone marrow-derived cells, endometrial stem cells, menstrual cells, neuroendocrine cells and intestinal cells.

Administration of Compositions

[0042] The compositions described herein can be introduced into a cell or administered to a subject (e.g., a human) in any suitable formulation by any suitable method. For example, one or more agents (e.g., mir-7) that regulate the expression of one or more miRNA target genes (e.g., SPATA2) may be injected directly into a cell, such as by microinjection. Alternatively, the molecules can be contacted with a cell, preferably aided by a delivery system. As another example, one or more agents (e.g., mir-7) that regulate the expression of one or more miRNA target genes (e.g., SPATA2) may be directly introduced into a mammal (e.g., human), including by intravenous (IV) injection, intraperitoneal (IP) injection, or in situ injection into target tissue (e.g., muscle). For example, a conventional syringe and needle can be used to inject a suspension containing one or more agents that regulate the expression of one or more miRNA target genes into a mammal. Depending on the desired route of administration, injection can be in situ (i.e., to a particular tissue or location on a tissue), IM, IV, IP, transcutaneous, or by another parenteral route. [0043] Any suitable delivery system can be used to administer a microRNA that modulates expression of a target gene involved in β-cell differentiation and/or replication/regeneration. Examples of delivery systems include liposomes, charged lipids, microspheres, protein transduction, dendrimers, biodegradable polymers, polymers of amino acids, ultrasound contrast agent delivery vehicles, etc. Methods which are well known to those skilled in the art can be used to construct a natural or synthetic matrix that provides support for the delivered agent (microRNA) prior to delivery. Matrices suitable for use in the invention may be formed from both natural or synthetic materials and may be designed to allow for sustained release of the therapeutic agent over prolonged periods of time. Various techniques using viral vectors (e.g., Adenoviral vectors, AAV vectors, lentiviral vectors, etc.) for the introduction of a microRNA into cells are provided for according to the invention. Viral vector methods and protocols are reviewed in Kay et al. Nature Medicine 7:33-40, 2001.

[0044] Ex vivo delivery of a composition including one or more agents that regulate the expression of one or more miRNA target genes is provided for within the invention. Islet- mediated ex vivo gene therapy may be used to transplant host islets expressing one or more agents that regulate the expression of one or more miRNA target genes back into the host pancreas. Any suitable delivery method may be used for transducing host islets expressing one or more agents that regulate the expression of one or more miRNA target genes ex vivo. Several suitable modes of delivery are described herein, including microinjection, electroporation, calcium phoshpate transfection, DEAD dextran transfection, polylysine conjugates, receptor-mediated uptake systems, liposomes, lipid-mediated delivery systems, intra-cellular targeting ligands, virion-like particles, and viruses. EXAMPLES

[0045] The present invention is further illustrated by the following specific examples.

The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1 - Mir-7 downregulates the SPATA2 gene and is differentially expressed during pancreatic islets development

[0046] The expression profile of miRNAs differentially expressed either in endocrine islets or in exocrine acinar tissue was investigated. Advances in microarray technology made possible the use of chip arrays to study the expression of miRNAs in various cells and tissues (Kim, V.N. & Nam, J. W. Genomics of microRNA. Trends Genet 22, 165-73 (2006)). Using this tool, a subset of miRNAs were identified that are preferentially expressed in pancreatic islets. First, miRNA expression profiles in rat islets versus acinar tissue were compared. Five miRNAs (mir-7, mir-183, mir-382, mir-127 and mir-29c) were identified having a zero probability value of being a false positive. Mir-7 has the highest rank, indicating the largest difference between the two groups: islet and acinar tissue (Fig. 1). To confirm the microarray results, selected islet miRNAs were analyzed by quantitative RT-PCR (qRT-PCR) utilizing a looped-primer RT-PCR method, which is particularly suited for the accurate and sensitive detection of miRNAs as well as other non-coding RNAs (ncRNAs). mir-375, which was not integrated in the Sanger mirBase release 8.1, was included in a qRT-PCR analysis. The results shown in Fig. 2 established that mir-7 is the most differentially expressed miRNA in islets. Its expression is more than 150-fold higher than in acinar tissue, while the fold-change ratio islet/acinar for mir-375 is approximately 7. A similarly high ratio of 250 (n=3 independent islet preparations) of mir-7 expression was observed in human islets as well. qRT-PCR analysis showed that mir-375 is 2.5 times more abundant than mir-7 in the islets (Fig. 2). Thus, mir-7 is the most differentially expressed miRNA in islets while mir-375 is the most abundant intraislet miRNA. This is in agreement with the frequency of cloning for mir-375 and mir-7, representing 8 and 4.3% respectively of the total number of clones obtained from pancreatic islets (Landgraf, P. et al. Cell 129, 1401-14 (2007)). The same study reports a high cloning frequency for mir-7 in the endocrine pituitary gland. The expression of mir-7 in islets and the pituitary gland represents more than 50% of the overall clones obtained from the screening of more than 26 organ systems (Landgraf, P. et al. Cell 129, 1401-14 (2007)). [0047] Collectively, these results indicate that mir-7 is an endocrine miRNA highly expressed in the islets of Langerhans. The sequence of mir-7 is conserved across vertebrates (Lim, L.P., Glasner, M.E., Yekta, S., Burge, CB. & Bartel, D.P. Vertebrate microRNA genes. Science 299, 1540 (2003)). mir-7 has been previously identified in zebrafϊsh islets (Wienholds, E. et al., MicroRNA expression in zebrafϊsh embryonic development. Science 309, 310-1 (2005)) and it was reported to promote photoreceptors in Drosophila (Li, X. & Carthew, R. W. A microRNA mediates EGF receptor signaling and promotes photoreceptor differentiation in the Drosophila eye. Cell 123, 1267-77 (2005)).

Expression of mir-7 in the developing mouse pancreas.

[0048] The mouse provides an unsurpassed model for mammalian pancreatic development (Murtaugh, L. C. Development 134, 427-38 (2007)). Between embryonic days e9.5 and -el 1.5, the first endocrine cells to appear express glucagon, followed by the first β- cells between el 1.5 and el 2.5. Around el 3.5 -el 4.5, the developing pancreas undergoes a process known as secondary transition, characterized by a large expansion of insulin- producing cells. By el 8.5 β-cell biogenesis declines and beta-cell expansion continues by replication. The expression of mir-7 and mir-375 was studied in pancreatic buds to determine if they are expressed postnatally and maintained during self-duplication or whether their expression originates during beta-cell biogenesis from pancreatic precursors. The expression profile of insulin 1 , insulin 2 and glucagon were also determined. Although mir-7 and mir- 375 showed dramatic increases in their expression during pancreatic development, their expression profiles are different (Fig. 3). The mir-7 expression profile precedes those of INSl and INS2 between el3.5 and el8.5, while the expression of mir-375 strictly correlates with that of insulin. As in mature islets, mir-375 is more abundant than mir-7 in all the stages with the exception of el4.5, in which both are equally expressed (Fig. 4). These results suggest a different role for mir-7 and mir-375 during β-cell biogenesis.

[0049] Matching of the miRNA sequence with corresponding mRNAs sequences allows for the prediction of target mRNAs for each miRNA. The RNA target predictive algorithm TargetScan (Grimson, A. et al. MoI Cell 27, 91-105 (2007)), identified the SPATA2 protein as one of the miRNA targets with the highest rank (Fig. 5). Referring to FIG. 5, Mir-7 binding sites in the 3'UTR of the SPATA2 gene are shown. Pairing usually occurs at the 3'UTR of a mRNA, and involves nucleotides 2-7 in the 5' domain of the miRNA, known as "seed" sequence (Lai, E.C. Nat Genet 30, 363-4 (2002); Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. & Burge, CB. Cell 115, 787-98 (2003)). The canonic complementary site is the 6mer involving nucleotides 2-7 from the seed sequence. Other recognized miRNA complementary sites are denominated 8mer, the 7mer-m8 and the 7mer-Al site (Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. & Burge, CB. Cell 115, 787-98 (2003); Lew, B.P., Burge, CB. & Bartel, D.P. Cell 120, 15-20 (2005); Brennecke, J., Stark, A., Russell, R.B. & Cohen, S.M. PLoS Biol 3, e85 (2005)). The 8mer site includes the 6mer match plus a match in the position 8 and the A at position 1 , the 7mer- m8 site includes the seed match plus a match in nucleotide 8 and the 7mer-Al contains the 6mer match plus the A match at position 1.

[0050] The SPATA 3'UTR contains three conserved sites (Fig. 5) and one poorly conserved site but still present in rat, mouse, human and dog transcripts. The contribution of all these sites puts SPATA2 in the highest percentile for mir-7 target sites by TargetScan algorithm and suggests the possibility that in the developing pancreas, mir-7 regulates the expression of SPATA2, perhaps contributing to the downregulation of its mRNA as shown for other target RNAs. The profile of SPATA2 expression was found to be inversely correlated with that of mir-7 throughout pancreatic development (Fig. 3). At the highest peak of mir-7 (el 8.5), the expression of SPATA2 decreases almost 50% compared to previous embryonic stages (Fig. 3).

Inhibition or overexpression of mir-7 inversely affects the steady-state level SPATA2 mRNA in insulinoma cells.

[0051] To investigate whether SPATA2 mRNA was indeed regulated by mir-7, overexpression and silencing in vitro protocols were used in cells naturally expressing SPATA2 such as insulinoma MIN6 cells. It was found that both treatments affected the steady-state level of SPATA2 mRNA. Morpholinos (MO) has been shown to inhibit the activity of mature miRNAs (Kloosterman, W.P., Lagendijk, A.K., Ketting, R.F., Moulton, J.D. & Plasterk, R.H. PLoS Biol 5, e203 (2007)). Treatment with anti-mir-7 MO directed against the two mature forms of mir-7 resulted in a significant increase of SPATA2 compared to cells treated with an irrelevant control MO (Fig. 6(a)). Likewise, transient overexpression of transfected pre -mir-7 RNA had a similar but reverse quantitative effect compared to cells treated with an irrelevant pre-mir RNA (Fig. 6(b)). This experiment strongly suggests that mir-7 is able to regulate the SPATA2 mRNA in cells expressing it naturally. The levels of glucagon remained unvaried in both treatments, while expression of insulin transcripts decreased with anti-mir-7 morpholinos and increased with pre-mir-7 RNA treatment. These results suggest an association between the expression of insulin, mir-7 and SPATA2. Gain and loss of function studies can be performed in targeted mice, both by in vivo overexpression and inhibition of mir-7, as well as overexpression and inhibition of putative target SPATA2.

[0052] The findings described herein are useful towards the development of new strategies to treat diabetes based on the generation of insulin-producing cells through β-cell replication, cell engineering and/or differentiation of their precursors.

Materials and Methods

[0053] Tissue procurement: Rat Islets were isolated from the pancreas of Lewis rats

(Charles River Labs) by digestion of the pancreatic tissue with purified enzyme blend Liberase RI (Roche Molecular Biochemical, Indinapolis, IN) at a concentration of 0.16mg/mL following the procedure described earlier (Embury, J. et al., Proteins linked to a protein transduction domain efficiently transduce pancreatic islets. Diabetes 50, 1706-1713 (2001)). Human pancreatic islets and acinar tissue were obtained from University of Miami, Diabetes Research Institute, Human Islet Cell Processing Facility. Pancreatic buds from embryos at gestation periods of el 1.5, el3.5, el4.5, el8.5 days (noon of the day a vaginal plug is found is considered 0.5 days of gestation) and from newborn CBA x B6 mice were isolated and microdissected as described previously (Fraker, CA. et al. Enhanced oxygenation promotes beta cell differentiation in vitro. Stem Cells (2007)). All animal studies were performed under protocols approved by the University of Miami Animal Care and Use Committee.

[0054] MicroRNA microarray studies: Total RNA was isolated by a method that preserves RNA molecules <200 bp, using mirVana miRNA Isolation kit (Ambion). Special caution was taken to prevent degradation of miRNAs and their cDNAs in RNA preparations in order to achieve comparative results for all the samples (Bravo, V., Rosero, S., Ricordi, C. & Pastori, R.L. Instability of miRNA and cDNAs derivatives in RNA preparations. Biochem Biophys Res Commun 353, 1052-5 (2007)). Samples were processed for miRNA profiling by LC Sciences (Houston, TX, USA). 2 to 5 μg total RNA sample was size fractionated using a YM- 100 Microcon centrifugal filter (from Millipore) to enrich the miRNA fraction. RNA was 3' extended with a poly(A) tail. An oligonucleotide tag was then ligated to the poly(A) tail for a subsequent fluorescent dye staining. Two different tags were used for the two RNA samples on each dual-sample chip (acinar and islet). Hybridization was performed overnight on a μParaFlo™ micro fluidic chip using a micro-circulation pump. The Array contained probes for 312 mature microRNAs (Sanger v. 8.1, June 2005) plus multiple controls including housekeeping small RNA genes and the oligonucleotide microRNA probes with single mutations (mismatch probe negative controls). Hybridization was carried out with 100 μL 6xSSPE buffer (0.90 M NaCl, 60 mM Na₂HPO₄, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 ⁰C.

[0055] After hybridization, fluorescence labeling with tag-specific Cy3 and Cy5 dyes provided detection. A total of 3 experiments (6 hybridizations) were performed using a "dye flip" where replicate hybridizations of the same sample and control were performed along with each dye direction. Hybridization images were collected with GenePix 4000B laser scanner (Molecular Devices, Sunnyvale, CA) and digitized by Array-Pro image analysis software (Media Cybernetics, Silver Spring, MD). Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally- weighted Regression) (Bolstad, B.M., Irizarry, R.A., Astrand, M. & Speed, T. P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185-193 (2003)). For two color experiments, the ratio of the two sets of detected signals (Iog2 transformed, balanced) and p-values of the t-test were calculated; differentially detected signals were those with less than 0.01 p-values.

[0056] Significant Analysis of Microarray (SAM) (Tusher, V.G., Tibshirani, R. & Chu,

G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98, 5116-21 (2001)) with False Discovery Rate (FDR)<5% was performed with all differentially expressed miRNAs with p-value <0.01 to identify significantly expressed genes across all replicate arrays. Cells cultures and tissues:

[0057] MIN6 insulinoma cells were cultured at 37 ⁰C with 5% CO₂ in DMEM medium supplememented with 55μM 2-mercaptoethanol, lOOμMsodium pyruvate and 10% fetal bovine serum (FBS).

[0058] miRNA quantification: qRT-PCR: RNA was isolated using the mirVana miRNA

Isolation kit (Ambion). Purity and concentration of the samples were assessed with a NanoDrop ND- 1000 Spectrophotometer. RQ of INSl, INS2, Glucagon, and SPATA2 was performed with 7500 Fast Real-Time PCR system, utilizing TaqMan Universal reagents and probes (Applied Biosystems, Foster City, CA). The quantification of miRNA was performed with a method termed looped-primer RT-PCR (Chen, C. et al. Nucleic Acids Res 33, el79 (2005)), following an Applied Biosystems protocol for TaqMan MicroRNA Assays. RT and PCR reactions were done in triplicate using looped-primers and primers specific for each miRNA (Applied Biosystems). RQ determines the change in expression of target transcripts in a test sample relative to a calibrator sample: either islets vs. acinar, or el3.5, el4.5, el8.5 and newborn pancreatic rudiments vs. el 1.5. (TaqMan miRNA assays panel). RQ was calculated via Applied Biosystems SDS software based on the equation RQ= 2^"ΔΔCt where Ct is the threshold cycle to detect fluorescence. Ct data were normalized to an endogenous control; 18S rRNA for mRNAs and mir-16 for miRNAs.

[0059] Mir-7 over expression: Pre-miR-7 precursor molecules and negative irrelevant control miRNA were transfected into MIN6 cells using siPORT NeoFX Transfection Agent from Ambion. The negative control anti-miRs were random sequence pre-miRs that had been tested in human cell lines and tissues and validated by the vendor (Ambion). Pre-miR molecules were diluted in optiMEM 1 medium to a final concentration of 5 nM, combined with the diluted transfection agent and then incubated at room temperature for 10 minutes. [0060] Anti-mir-7 morpholinos treatment: Anti-mir-7 or irrelevant morpholinos (Gene

Tools, Philomath, OR,US A) were dissolved to a concentration of lOOμM. Cells were incubated for 48 hs at lOOμM of each morpholinos and X μl/ml of culture of Endoporter (Gene Tools) delivery agent. At the end of the incubation the cells were washed and RNA isolated as described above.

Example 2 - MicroRNA mir-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas

[0061] MicroRNAs (miRNA) have been predicted to target genes important for pancreas development, proper endocrine cell function and metabolism. As described above, miRNA-7 (miR-7) was the most abundant and differentially expressed islet miRNA, with 200-fold higher expression in mature human islets than in acinar tissue. To elucidate a potential role for mir-7 during pancreatic development, the temporal and spatial expression of mir-7 in human fetal pancreas from 8 to 22 wga of gestational age (wga) was examined. Human fetal (8-22 wga) and adult pancreases were processed for immunohistochemistry, in situ hybridization and quantitative RT-PCR of miRNA and mRNA.

[0062] The expression of miR-7 in adult and fetal tissue, from 8 to 22 weeks of gestational age (wga), and adult human pancreas was first examined by quantitative real-time PCR (qRT-PCR) (Fig. 7). MiR-7 expression was very low between 8 to 12 wga. At week 13 the expression of miR-7 started to increase and by week 14 a 45 -fold increase in expression, compared to the level at 8 wga, was observed. Moreover, the increase in miR-7 expression coincided with the time at which expression of hormone encoding genes began to dramatically rise (Fig. 7A). MiR-7 levels remained high until 18 wga to decrease afterwards. The expression of mir-127 and mir-382, two miRNAs shown to be preferentially expressed in adult islets, and mir-375 showed a similar pattern of expression (Fig. 7). Also, the period with higher expression of miR-7 (14-18 wga) corresponded with the maximum expression levels of the pro-endocrine marker NGN3, albeit with a slightly delayed profile (Fig. 7A). [0063] Next, miR-7 expression was analyzed during human pancreas development and in normal adult human pancreatic tissue by in situ hybridization using an anti-miR-7 LNA probe (Fig. 7B). LNA probes are bi-cyclic RNA analogs that allow an enhanced stringency for short probes as required for miRNA detection (Petersen et al., J MoI Recognit., 13:44-53, 2000; Kloosterman et al., Nat Methods 3: 27-29, 2006). MiR-7 expression was first observed in the epithelium of 9 wga fetal pancreas. The number of miR-7 expressing cells was very low from 9 to 11 wga but increased, concomitantly with an increased intensity of expression, to form clusters of intensely stained miR-7-expressing epithelial cells by week 14 onwards. MiR-7 expression levels at 14 and 17 wga appear similar even though there is a decline in the relative expression as detected by RT-PCR after 14 wga. This is however not surprising since the relative amount of non-endocrine tissue increase during this period, thus "diluting" the relative miR-7 expression values obtained by RT-PCR analyses.

[0064] On a morphological level the in situ hybridization revealed that miR-7 was expressed in the epithelial part of the developing pancreas. In order to identify the nature of the cells expressing miR-7, counterstaining of the in situ hybridized sections with antibodies specific for markers of endocrine precursors and differentiated endocrine cells was performed. The 14 wga human fetal pancreas was selected since it showed the highest relative miR-7 expression by RT-PCR. Hormones levels dramatically rise and islet-like structure start to appear at this stage (Fig. 7).

[0065] The expression of IPF1/PDX1, a homeodomain transcription factor expressed in early pancreatic progenitor cells and in differentiated beta cells, was detected in mir-7 positives cells (Fig. 8). However, some of the cells present in mir-7-positive clusters did not express IPFl suggesting that these cells could be non-beta endocrine cells (Fig. 8). Likewise, many IPFl -positive cells did not express mir-7, probably because they were undifferentiated precursors cells. Next, the expression of mir-7 was compared with that of ISL-I, a marker for differentiated endocrine cells, and these analyses showed that most of the mir-7 cells co- expressed ISL-I. However, and as expected, the vast majority of cells expressing ISL-I did not express the pro-endocrine gene NGN3 (Fig. 8). The expression of mir-7 and NGN3 were thus indirectly compared based on the co-expression of mir-7 and NGN3, respectively, with that of ISLl. Taken together, these data strongly suggests that NGN3 and mir-7 are not co- expressed. Furthermore, the mir-7 expression profile is slightly delayed from that of NGN3 (Fig. 7A) and all insulin, glucagon and somatostatin positive cells expressed mir-7 in fetal and adult tissue (Fig. 8 and Fig. 9). In adult pancreatic tissue, the expression of mir-7 was clearly restricted to the islet cells; ductal (CKl 9 positive) and acinar (amylase positive) cells were negative for mir-7 (Fig. 9).

[0066] The findings described herein show that mir-7 is predominantly expressed in endocrine cells in the developing and adult human pancreas, and confirm an endocrine- specific expression for mir-7 in the adult pancreas and also show an endocrine-restricted expression in the developing human pancreas. These results suggest that mir-7 is a marker of differentiated endocrine cells and that mir-7 expression appears in the differentiating endocrine cells following the transient expression of NGN3.

Experimental Procedures

[0067] Human fetal pancreases from 8 to 22 wga were collected from fetal tissue immediately after elective termination of pregnancy from healthy women that were admitted to local clinics and gave their proper consent to use fetal tissues for research studies. The study is in compliance with US legislation and the guidelines of the University of Miami. Gestational age was determined on the basis of time since the last menstrual period, the measured of the Crown-Rump Length (CRL), and the biparietal diameter (BPD) by ultrasonography. Human adult pancreas fragments were obtained from the Human Islet Cell Processing Facility at the Diabetes Research Institute. Two or more independent pancreases were analyzed in each stage.

[0068] MiRNAs were quantified from total RNA isolated using mirVana miRNA

Isolation kit (Ambion-Applied Biosystems, Foster City, CA) and mRNAs were quantified using total RNA isolated using RNeasy Mini kit (Qiagen) according to the manufacturer's directions. Purity and concentration of the samples were assessed with a NanoDrop ND- 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). The quantification of miRNA was performed with a method termed looped-primer RT-PCR (Chen et al., Nucleic Acids Res. 33:el79, 2005), following the Applied Biosystems protocol for TaqMan MicroRNA Assays and the quantification of mRNA was carry out using FAM labeled target gene probe (Applied Biosystems) in a 7500 Fast Real-Time PCR system. For a particular sample, values are shown as RQ (relative quantification), the ratio of expression of a given miRNA or mRNA over the expression of the endogenous 18S ribosomal RNA. RT and PCR reactions were performed in triplicates. RQ was calculated via Applied Biosystems SDS software based on the equation RQ= 2^"ΔΔCt where Ct is the number of cycles at which amplification reaches a threshold, determined by the software, within the exponential amplification phase.

[0069] In situ hybridization using fluorescein-labeled anti-human miR-7 Locked

Nucleic Acid (LNA) probe (Exiqon, Denmark) or DIG-labeled NGN3 probe was performed essentially as described (Obernosterer et al., Nat Protoc 2:1508-1514, 2007). A total of 1 pmol of fluorescein-labeled LNA or 30ng of DIG-labeled NGN3 probe were diluted into 150 μl of hybridization buffer, applied to the slides and allowed to hybridize at 60⁰C or 70⁰C respectively overnight. Slides were then washed for 1 h in 0.2 x SSC solution (Ambion- Applied Biosystems, CA) and incubated with alkaline phosphatase-conjugated sheep anti- fluorescein antibody (1/1500, Roche) or alkaline phosphatase-conjugated sheep anti-DIG (1/2500, Roche) antibodies overnight at 4°C. Alkaline phosphatase reaction was carried out in PVA with 200μl of MgCl₂ IM and 140μl of NBT/BCIP stock (Roche) for 1-3 days. Hybridization with sense fluorescein-labeled LNA miR-159 (Exiqon) was used as control. MiR-159, a miRNA described in Arabidopsis thaliana does not have a known target in the Sanger miRBase. Human fetal intestine and spleen, tissues in which miR-7 was undetectable by RT-PCR, were used as negative control and also negative control assays were performed without primary antibodies.

[0070] The primary antibodies and the dilutions used were: guinea pig anti human-

Insulin (1/50, Dako), mouse anti-glucagon (1/1500, Sigma), anti-somatostatin (1/500, Dako), mouse anti-Cytokeratin 19 (1/50, Biogenex), rabbit anti- Amylase (1/200, Sigma) rabbit anti- IPFl (1/4000), generated against human IPFl peptide by Agrisera AB) and rabbit anti-ISLl (1/250, (Ahlgren et al., 1997)). Secondary antibodies used were: Alexa Fluor 488-conjugated goat anti-rabbit (1/400, Molecular Probes), Alexa Fluor 568-conjugated goat anti-guinea pig (1/400, Molecular Probes) and Alexa Fluor 647 Goat anti-mouse (1/400, Molecular Probes). DAPI (12.5μg/ml, Invitrogen) was used as a nuclear counterstaining. [0071] Results are expressed as arbitrary units and are presented as the mean value ± standard errors (S. E. M)

Example 3 - Inhibiting insulin formation by modulating mir-7 expression in vivo [0072] To define the role of mir-7 in endocrine pancreas formation and/or function, loss of function studies were performed by inhibiting mir-7 activity in the fetal pancreas in vivo. It was hypothesized that manipulation of mir-7 will identify genes regulated by this miRNA that might be important to islet cells function. Inhibition of mir-7 is achieved by transfecting specific antisense morpholinos (MO) into the cells. MOs are synthetic antisense oligos that mimic DNA. They can block miRNA maturation at the step of the Drosha or Dicer cleavage, two critical proteins involved in the maturation of miRNAs. MOs can also inhibit the activity of the mature miRNA. MO oligos for miRNA blockade were obtained from Gene Tools (LLC, Philomath, Oregon). To investigate if delivery of MO via intra- fetal heart would reach the pancreas, a FITC-labeled MO (MO-FITC) directed against irrelevant sequence was injected at elθ.5, the pancreatic bud was isolated the next day and analyzed by confocal microscopy. The fluorescence pattern clearly indicated a good penetration of MO throughout the tissue structure. It was concluded that it is possible to deliver MO intra-fetus to block mir- 7.

[0073] In the mouse model, between embryonic days e9.5 and el 1.5, the first endocrine cells to appear are alpha cells expressing glucagon, followed by the first insulin-producing β cells between el 1.5 and el2.5. Around el3.5-el4.5, the developing pancreas starts a process known as secondary transition, characterized by a large expansion of insulin-producing cells. Therefore, MO anti-mir-7 or irrelevant MO was injected into the fetal heart at elθ.5 to study if manipulation of mir-7 has an effect on insulin expression (Fig. 10). Following injection, pancreatic buds were retrieved at el 4.5. MO anti mir-7 but not irrelevant MO inhibited the formation of insulin in the fetal pancreas (Fig. 10).

[0026] These experiments suggest that inhibition of mir-7 could have a critical role in the function/development of β cells.

Other Embodiments

[0074] Any improvement may be made in part or all of the compositions, systems, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

Claims

What is claimed is:

1. A composition consisting of a pharmaceutically acceptable carrier and a therapeutically effective amount of a miRNA that modulates expression of at least one miRNA target gene in a cell for inducing at least one of: differentiation of non-insulin- producing cells into insulin-producing cells, proliferation of insulin-producing cells, and regeneration of insulin-producing cells.

2. The composition of claim 1 , wherein the at least one miRNA target gene is selected from the group consisting of: SPATA2, Zeb2, SOX4, and CDKNBlB.

3. The composition of claim 1 , wherein the miRNA is mir-7 miRNA.

4. The composition of claim 3, wherein the mir-7 miRNA is comprised within a vector.

5. A method of inducing insulin-producing cell proliferation in vitro, the method comprising the steps of: a. providing a composition consisting of a pharmaceutically acceptable carrier and an effective amount of a miRNA that modulates expression of at least one miRNA target gene in a cell for inducing at least one of: differentiation of non-insulin-producing cells into insulin-producing cells, proliferation of insulin-producing cells, and regeneration of insulin-producing cells; b. contacting a plurality of insulin-producing cells with the composition; and c. culturing the plurality of insulin-producing cells under conditions in which they proliferate.

6. The method of claim 5, wherein the at least one miRNA target gene is selected from the group consisting of: SPATA2, Zeb2, SOX4, and CDKNBlB.

7. The method of claim 5, wherein the miRNA is mir-7 miRNA.

8. The method of claim 7, wherein the mir-7 miRNA is comprised within a vector.

9. A method of inducing differentiation of non-insulin-producing cells into insulin-producing cells in vitro, the method comprising the steps of: d. providing a composition consisting of a pharmaceutically acceptable carrier and an effective amount of a miRNA that modulates expression of at least one miRNA target gene in a cell for inducing at least one of: differentiation of non-insulin-producing cells into insulin-producing cells, proliferation of insulin-producing cells, and regeneration of insulin-producing cells; e. contacting a plurality of non-insulin-producing cells with the composition; and f. culturing the plurality of non-insulin-producing cells under conditions in which they differentiate into insulin-producing cells.

10. The method of claim 9, wherein the at least one miRNA target gene is selected from the group consisting of: SPATA2, Zeb2, SOX4, and CDKNBlB.

11. The method of claim 9, wherein the miRNA is mir-7 miRNA.

12. The method of claim 11 , wherein the mir-7 miRNA is comprised within a vector.

13. A method of modulating mir-7 microRNA expression in a mammal, the method comprising the steps of: a. providing a composition consisting of a therapeutically effective amount of an agent that increases or decreases expression of mir-7 miRNA expression and a pharmaceutically acceptable carrier; and b. administering the composition to the mammal, wherein administration of the composition to the mammal modulates mir-7 microRNA expression in the mammal.

14. The method of claim 13, wherein the composition is administered to the pancreas of the mammal.

15. The method of claim 14, wherein the composition is administered to the mammal by injection.

16. A method of modulating insulin expression in a mammal, the method comprising the steps of: a. providing a composition consisting of a therapeutically effective amount of mir-7 miRNA and a pharmaceutically acceptable carrier; and b. administering the composition to the mammal, wherein administration of the composition to the mammal modulates insulin expression in the mammal.

17. The method of claim 16, wherein the composition is administered to the pancreas of the mammal.

18. The method of claim 17, wherein the composition is administered to the mammal by injection.

19. The method of claim 16, wherein the mir-7 miRNA is comprised within a vector.

20. The method of claim 16, wherein the mir-7 miRNA further modulates the expression of a target gene selected from the group consisting of: SPATA2, Zeb2, SOX4, and CDKNBlB.